University Of Illinois At Chicago

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT - MECHANOSURVEILLANCE IN BREAST CANCER METASTASIS Metastatic breast cancer is a devastating disease that impacts the lives of tens of thousands of women each year. Unfortunately, the majority of treatment options for patients diagnosed with metastatic breast cancer are palliative. Current treatment strategies often target cancer cells’ genetic and biochemical abnormalities, but cancer cells also undergo profound changes in their biophysical properties such as cellular stiffness. Targeting biophysical properties offers exciting new therapeutic avenues, but the basic knowledge on the relationship between cancer cell stiffness and regulation of metastasis is incomplete. Emerging evidence suggests that cancer cell stiffness acts as a mechanical input for activation of cytotoxic lymphocytes that destroy cancer cells. Thereby, stiffness becomes an immune vulnerability of cancer cells during the metastatic cascade. This process, where mechanical inputs activate the immune surveillance, is termed mechanosurveillance and it is strongly regulated by the expression of Myocardin related transcription factors A and B (MRTFA/B) in cancer cells. Based on the published and the preliminary data, the overarching hypothesis of this project is that MRTFA driven calcium influx increases cancer cell stiffness and thereby activates mechanosurveillance during breast cancer metastasis. The long-term goal of this project is to determine the molecular mediators of MRTFA associated mechanosurveillance and thereby inform pharmacological methods to exploit this cancer cell vulnerability for better treatment of patients with metastatic breast cancer. The research design includes a set of interdisciplinary approaches, such as transcriptomic and genomic analyses of patient samples and cancer models, atomic force microscopy on patient derived xenograft organoids (PDXos) and on immunocompetent models of metastatic colonization, and histological analyses of human breast cancer metastases. The specific aims of this project are: 1) to determine the contribution of MRTFA driven calcium influx on cancer cell stiffness in PDXos, in vitro, and in situ mouse models by using atomic force microscopy and confocal microscopy. 2) to determine the degree of cytotoxic lymphocyte activation by MRTFA and the associated stiffness in vivo by using multiplexed imaging and machine learning on metastatic tissue samples from patients and by using flow cytometry in metastases in syngeneic mouse models. 3) to determine how to exploit MRTFA associated mechanosurveillance to target metastases by testing the impact of clinically relevant immune checkpoint blockade treatments on metastasis bearing mice. This work will uncover mediators of mechanosurveillance and demonstrate its prevalence in human cancer. Thus, results of this work will impact the immediate use of immune checkpoint blockade and the future strategies to enhance mechanosurveillance in patients with metastatic breast cancer.

NIH Research Projects · FY 2026 · 2022-09

The autophagic and lysosomal pathways (ALP) clear misfolded proteins and damaged organelles from cells. Their function is therefore particularly critical for long-lived cells such as neurons. Dysfunction in the ALP is associated with various stages of Alzheimer’s disease (AD). This includes the robust accumulation of autophagosomes and lysosome-like organelles in dystrophic axons around extracellular Aβ deposits (Amyloid plaques), which are hallmark pathological features observed in human Alzheimer’s disease brain tissue and recapitulated in transgenic mouse models of Alzheimer’s disease. Amyloid plaque formation has been directly linked to aberrant/amyloidogenic proteolytic processing of amyloid precursor protein (APP) by secretases. However, whether abnormal trafficking and accumulation of these organelles bearing these protein cargoes trigger this critical pathogenic event in Alzheimer’s disease, has not been experimentally addressed. In addition, information on mechanisms and specific molecular components regulating ALP in axons remains limited. Elucidating these mechanisms and identifying molecular components might enable therapeutic modulation of neuronal ALP to reduce amyloid plaque burden and toxic Aβ peptide production in Alzheimer’s disease. To this end, our proposed research seeks to understand a) how the adaptor complex, AP-4, regulates axonal autophagosome and lysosome biogenesis, maturation, and transport; b) how loss of AP-4 contributes to amyloid plaque formation in vivo as well as potentially identify new AP-4 cargo that facilitate optimal retrograde axonal lysosome transport, APOE metabolism, and synaptic activity. Central to these proposed studies is our preliminary data that AP-4 loss causes abnormal accumulation of ALP organelles in axonal swellings reminiscent of AD pathology, including build-up of APP cleaving proteins BACE1 and PSEN2. This and our preliminary data demonstrating reduced AP-4 levels in AD mouse brain and an age-dependent loss of AP-4 in the pre-frontal cortex of even wild type mice lead us to hypothesize that AP-4-dependent axonal autophagosome and lysosome maturation and transport protects neurons from amyloidogenic APP processing and thus, from amyloid plaque development. Additionally, our use of super-resolution microscopy on ALP organelles in axonal swellings as well as proteomics on isolated axons and organelles upon loss of AP-4 complex, will yield novel insight into the kinds of ALP intermediates accumulating under these pathological conditions and their relative contributions to build- up of APP processing machinery. Our proposed efforts to dissect out AP-4 mediated axonal ALP transport and maturation could also lead to new therapeutic opportunities that focus on mobilizing these axonal ALP organelles to limit Aβ production and axonal pathology. Furthermore, new insights into cell biology of neuronal autophagic and lysosomal pathways revealed by our studies could have broader relevance to other neurodegenerative diseases that have a lysosome component to their pathology such as Parkinson’s disease, Hereditary Spastic Paraplegia, and fronto-temporal dementia.

NIH Research Projects · FY 2026 · 2022-09

Down Syndrome (DS) is a genetic disorder caused by the triplication of chromosome 21. DS is characterized by defective brain development, manifested by intellectual disability. The molecular mechanisms causing it are not fully understood. Amyloid precursor protein (APP) resides on chromosome 21, thus triplicated in DS. APP plays a role in developmental and post-natal neurogenesis. However, studies examining the role of APP overdose on cortical malformation in DS are scarce. Approximately fifty percent of persons born with DS develop Alzheimer’s Disease (AD) by sixty years of age. Mutations in APP cause familial AD. Increased dosage of APP is predicted to result in enhanced accumulation of APP cleavage products including β-amyloid (Aβ). Aggregated Aβ comprises hallmark amyloid plaques observed in AD brains. Notably, patients with trisomy of chromosome 21 lacking the APP locus do not develop AD. Thus, it would be important to determine whether an additional copy of APP is essential or necessary for the development of AD in DS. Our preliminary studies show that signals critical for neural stem cell proliferation, neuronal differentiation and lamina specification are reduced in DS organoids. This project will test the hypothesis that APP overexpression causes or contributes to deficits in neurogenesis and Alzheimer’s pathology in Down Syndrome. We applied CRISPR-Cas9 technology to eliminate one copy of APP from Down syndrome-derived iPSC [DSAPP]. Using brain organoids developed from DSAPP, DS and isogenic iPSC, experiments in Aim 1 will examine the role of APP in neural stem cell proliferation and neural lineage commitment in DS, Aim 2 will address the role of APP in neuronal differentiation and cortical organization, and Aim 3 will examine whether APP overdose in DS is essential or necessary for the development of AD pathology. These experiments will address critical gaps in understanding DS and AD and identify new therapeutic targets for these disorders.

NIH Research Projects · FY 2025 · 2022-09

Chronic kidney disease (CKD) is a major public health problem in the United States that affects 37 million Americans and disproportionately burdens racial and ethnic minority populations. Advances in personalized medicine approaches for patients with CKD lag behind advances in other fields. Current approaches to classifying CKD do not provide granular insight into the underlying mechanisms that contribute to the heterogeneity of the disease. To address this gap, NIDDK established the Kidney Precision Medicine Project (KPMP) in 2016 with the overarching goal of using deep molecular phenotypes of kidney biopsies, along with longitudinal clinical phenotypic data to develop new disease ontologies and treatments for kidney disease. This proposal for a University of Illinois at Chicago (UIC) KPMP CKD Recruitment Site brings together a diverse multidisciplinary team of experienced investigators with expertise in nephrology, renal pathology, interventional radiology, bioethics, social determinants of health, and biostatistics. Our team has a successful track record in recruiting and retaining large and diverse populations of patients with CKD in long-term studies. This proposal has three overarching goals: 1) Utilize our well-established and successful recruitment strategies to enroll 100 participants (50% non-Hispanic Black, 30% Hispanic) from the University of Illinois Health Science System and a large affiliated federally qualified health center to undergo high-quality research kidney biopsies, conforming to the highest ethical, research and clinical standards; 2) Leverage the infrastructure of the UIC Chronic Renal Insufficiency Cohort (CRIC) Study Clinical Center to enroll 50 eligible CRIC participants into KPMP, providing an unprecedented opportunity to link data from this deeply phenotyped cohort with KPMP kidney biopsy measures; and 3) Implement a systematic assessment of neighborhood-level measures of social determinants of health which will advance the overall mission of KPMP to better characterize disease subgroups. We will use an established real-time electronic health record-based reporting system to identify eligible individuals in the two health care systems and then engage potential participants in an open discussion of the risks and benefits of a research kidney biopsy. Additionally, we will utilize patient-centered strategies to maintain long- term patient engagement and achieve high rates of retention with the guidance of a local Community Advisory Board composed of key stakeholders (i.e., patients, a caretaker, and a primary care provider). Leveraging our expertise and experience working with NIDDK U01 cooperative projects, we will rapidly and effectively implement the KPMP protocol, work collaboratively with the consortium, and will make substantial contributions to the long-term scientific mission of KPMP to develop precision medicine-based approaches for treating kidney disease.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Cancer has claimed over 600,000 lives in 2020 in the United States. A better understanding of the mechanisms underlying cancer progression has led to the development of early detection strategies and novel treatment modalities that have contributed to the decrease in cancer-related deaths observed for the past few decades. Yet, cancer remains a deadly disease. There is thus an acute need to identify new cancer vulnerabilities. This will require exploring understudied aspects of cancers, which requires the development of novel technologies. One understudied aspect of cancer is the extracellular matrix (ECM). The ECM is a complex meshwork of proteins providing architectural support and biochemical signals critical for cellular functions required for tumor progression. Overcoming technical challenges posed by largely insoluble ECM proteins, we previously devised a proteomic pipeline specifically geared towards ECM proteins and showed that the tumor ECM is composed of 200+ distinct proteins. We further identified ECM signatures predictive of patient outcome and novel ECM proteins playing functional roles in cancer progression. The ECM thus represents an important reservoir of potential prognostic biomarkers and therapeutic targets. However, the ECM has many more secrets to reveal. For example, ECM proteins exist in various isoforms and are extensively post-translationally modified, yet, we do not know which proteoforms are present in the tumor ECM. ECM protein structure and the architecture of the ECM meshwork is key to mediate function, yet, very little is known about ECM protein folding and its impact on protein functions. Since proteomics relies on the generation of peptides from protein via proteolysis and protein identification via database search, we propose that enhancing these steps will provide a more complete picture of the cancer ECM and significantly advance cancer research. Here, we propose to use in-silico modeling to define the optimal cleavage conditions to achieve near-complete coverage of ECM protein sequences (Aim 1). Standard proteomic protocols rely on protein denaturation prior to protein digestion. Yet, we know that many ECM functions are governed by its architecture. We thus propose to perform native ECM digestion to gain insights into the structure of individual proteins, and the secondary and tertiary structures of the ECM meshwork (Aim 2). To facilitate ECM research, we have previously developed a searchable database, MatrisomeDB, compiling ECM proteomic dataset. Here, we propose to enhance the content and functionalities of MatrisomeDB to include our new prediction model and a new tool to the visualize sequence coverage on 3D models of ECM proteins predicted by Google’s AlphaFold (Aim 3). Our technology, offering substantial improvements over conventional proteomic approaches, targets the unmet technical need to profile, with deep coverage and high sensitivity, the protein composition of the tumor ECM. When deployed it will significantly lower the technical barrier for other researchers to study the ECM, which will have a transformative impact on cancer research.

NIH Research Projects · FY 2025 · 2022-09

There has been an unprecedented increase in the number of refugees worldwide, with approximately 26 million men, women and children forcibly displaced from their homes at the end of 2019. More than half of the world’s refugees live in dense urban areas or refugee camps in low- and middle-income countries. A smaller percentage are permanently resettled in high-income countries. Across these contexts, refugees have elevated rates of common mental disorders. War and forced migration also contribute to systematic disruptions in social relationships including family separation, tension and conflict, and losses of social networks. There is an emerging evidence base of models regarding refugees’ mental health and psychosocial needs, but major gaps exist regarding how to adapt evidence-based interventions (EBIs) to refugee populations and sustain them in community settings. To address these gaps, my career goal is to establish an independent research program focused on the implementation of community-based mental health services for refugee communities across the displacement continuum. In collaboration with community partners, I aim to examine methods for integrating EBIs into non-traditional service settings to enable refugees’ access to prevention and care interventions that promote positive mental health outcomes. A central concern my research seeks to address is how to best mobilize social and family resources to enhance coping and wellbeing. These research priorities are shaped by my more than fifteen years of clinical practice experience working with war-affected populations in the U.S and globally, doctoral training at the University of Chicago and early research experiences. This K01 application is structured to build upon my strengths and develop new knowledge and skills in key areas needed to achieve my long-term goals. My training aims focus on 1) implementation science methods to adapt, scale up and sustain EBIs in community settings; 2) the application of human-centered design to community-based refugee mental health services; and 3) community-based trial design and analysis. With support from an accomplished multidisciplinary team of mentors, I will apply these skills to a research project that focuses on implementing a peer-led multiple family group (MFG) prevention intervention called CAFES in two community-based organizations in Chicago to promote uptake and meet the multi-level needs of refugee families. Using a pilot randomized type 1 hybrid implementation-effectiveness design, the research aims include: 1) Adapting the multiple family CAFES model for Syrian refugee families and delivery by peer providers in community-based organizations using the ADAPT-ITT framework and human-centered design methods; 2) Piloting the adapted CAFES model to assess implementation science domains; and 3) Exploring changes in mental health outcomes and family and community support and mechanisms of change. Drawing on the new knowledge and skills and pilot study data obtained from this K01, I will prepare an R01 proposal to further test the model implementation and effectiveness.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Our laboratory is interested in understanding how eukaryotic cells ensure the maintenance of telomeres, the natural ends of linear eukaryotic chromosomes. Evolutionarily conserved shelterin and CST (CTC1/Cdc13- STN1-TEN1) complexes play essential roles in telomerase recruitment and protection of telomeres against DNA repair and checkpoint factors. Stable maintenance of telomeres is critical to preserve genomic integrity and prevent accumulation of undesired mutations that might lead to tumor formation. Regulation of telomere structures and telomerase also affect cell proliferation and tissue maintenance in aging organisms. Therefore, basic mechanistic studies investigating how telomere and DNA damage response proteins collaborate in proper telomere maintenance should provide critical insights necessary to help devise more effective treatment strategies against tumors or other age-related diseases. Our proposed research projects utilize fission yeast Schizosaccharomyces pombe. Fission yeast telomeres serve as a good model for human telomeres, since proteins involved in telomere maintenance are highly conserved between fission yeast and humans. Studies from our lab and others have provided detailed insights how fission yeast shelterin and Stn1-Ten1 ensure stable maintenance of telomeres in fission yeast. Those include findings that (1) Tel1ATM/Rad3ATR_ dependent phosphorylation of the shelterin subunit Ccq1 on Thr93 promotes telomerase recruitment by promoting interaction between Ccq1 and the telomerase subunit Est1, and (2) SUMOylation of another shelterin subunit Tpz1ree, on Lys242 facilitates Stn1-Ten1 recruitment to telomeres and limits telomere extension. Evolutionarily conserved "TEL patch" residues within Tpz1 have also been found to promote telomerase activation and recruitment, further highlighting the well conserved nature of telomere regulation by fission yeast and mammalian shelterin. Our analyses of temporal binding patterns for DNA polymerases, telomerase, shelterin and Stn1 found that shelterin subunits Rap1 and Poz1 and the Stn1-Ten1 complex promote timely dissociation of telomerase from telomeres by promoting recruitment of Pola to complete lagging strand synthesis at telomeres. For the current grant application, our proposed experiments will (1) identify and characterize underlying regulatory mechanism(s) that allow Ccq1 and Poz1 to promote Pola-dependent telomere protection (Aim 1), (2) identify new interaction partners of Stn1-Ten1 complex and characterize their contributions to recruitment/retention of Stn1-Ten1 complex at telomeres and non-telomeric sites (Aim 2), and (3) investigate how regulation of TERRA vs. poly(A)+ TERRA expression modulates Stn1-Ten1-Pola recruitment at telomeres (Aim 3).

NIH Research Projects · FY 2025 · 2022-09

GLCFHW Overall Center Abstract Hired migrant and seasonal farmworkers (FWs) experience unique employment conditions that challenge their ability to live healthy, fulfilled lives. Hazardous work, unstable employment, atypical employment policies, lack of government oversight and enforcement, extreme poverty, stringent immigration laws, and lack of social support negatively impact the health and well-being of FWs. Further impediments include lengthy journeys across dangerous borders, inconsistent immigration policies, transience, non-citizen status, residence in remote rural settings, and limited access to healthcare and social services. Illinois is a major agricultural state that hires upward of 50,000 FWs each year to support the US food supply chain. The overall goal of the Great Lakes Center for Farmworker Health and Well-being (GLCFHW) is to implement strategies and actions leading to systems change that protects and promotes the health and well-being of farmworkers across the US. Specific aims are to: 1) conduct high quality research that elucidates pathways for change that benefits FWs; 2) build and strengthen multidirectional collaboration and engagement across eco-social levels to create networks that support FW health and well-being; 3) translate and disseminate evidence to promote policies and practices that demonstrate the value and approaches to supporting FW health and well-being within and outside of work. We have assembled Center Administration, cores in Evaluation and Planning, Outreach, and Research, and Internal and External Advisory groups and we have garnered the support of stakeholders. We will conduct three research projects: 1) to translate and test survey tools; 2) to link state-based data systems and outpatient records for surveillance of illness and injury among FWs; 3) to collect survey data on lived experience and biospecimens for markers of inflammation and immune response to determine predictors of health and well-being. We will disseminate research results and translate our findings to action. Outputs. validated survey tools, surveillance data, methods and findings from research studies, network analysis, environmental scan, strategic plan, outreach and evaluation protocols, website w/ data visualization. Outcomes. an established center focused on farmworkers, evidence for interventions, network of researchers and multilevel collaborators, pathways and mechanisms for dissemination, translation of findings to action NIOSH Objectives. NORA: Reduce risk of fatal/nonfatal injuries and work-related illnesses to workers and vulnerable populations in the agriculture sub-sector. Improve reporting and surveillance. Identify and examine the impact of worker demographics on employer or organizational practices and worker safety, health, and well-being. Improve the safety, health, and well-being of workers with non-standard work arrangements. Other alignments: research to practice (r2p), Total Worker Health promotion/protection of worker health and well- being; translation and validation of NIOSH Worker Well-being Questionnaire in precarious worker population.

NIH Research Projects · FY 2024 · 2022-09

Falls are the most common cause for injury-related hospitalization in older adults and account for over $50 billion per year in medical costs. For the ~27% of US adults with symptomatic osteoarthritis (OA) the odds of falling are up to 54% higher than for people without OA. The high incidence of falls associated with OA remains high after total knee arthroplasty (TKA). The incidence of falls during the first year after TKA is up to 51.8%. With over 750,000 TKAs performed annually, the scope of the problem is large. Despite several calls in the literature for new methods to decrease fall rate after TKA, there are, to our knowledge, no such interventions specifically targeting this group. Our recent work, however, demonstrates the promise of perturbation training in knee OA. We have established that limiting trunk angle and angular velocity during the initial recovery step after a disturbance, such as a trip, is a critical factor in avoiding a fall. This motor skill can be improved through task-specific perturbation training. In our pilot study, trip-specific perturbation training effectively improved trunk kinematics during the initial recovery step after a trip-simulating perturbation in women with moderately symptomatic OA. Further, we have shown that trip-specific perturbation-training reduces fall-rates in the community in healthy older women. Building on this past success, our long-term goal is to reduce the incidence of preventable gait-related falls by older adults with joint replacements. The objective of this application is to establish that preoperative task-specific training of stepping responses to sagittal and frontal plane disturbances reduces the risk of postoperative falls in people undergoing TKA. Our central hypothesis is that individualized preoperative sagittal and frontal plane perturbation training will significantly reduce postoperative fall rates in people scheduled for TKA by improving trunk kinematics during the initial recovery step. To test this hypothesis, we will evaluate preoperative TKA patients, randomized into either a perturbation-training group or a conventional fall-prevention education group. We will assess our central hypothesis through two aims. Aim 1: Demonstrate the extent to which perturbation training focused on forward and laterally directed stepping responses improves recovery step trunk mechanics in preoperative TKA patients. Aim 2: Demonstrate the effectiveness of forward and laterally directed perturbation training in reducing incidence of preventable gait- related falls for one year after TKA. With extensive collective expertise in OA and arthroplasty gait mechanics, aging, falls, and fall-prevention, including clinical trial experience, we are well poised to accomplish these aims. Further, through our innovative prehabilitation approach, we will, for the first time, address both trip-specific and laterally directed falls in this patient population. In addition, this study will advance our mechanistic understanding of trip-specific and laterally directed fall risk in people before and after TKA. By introducing a new prehabilitation approach to reduce postoperative falls in people undergoing TKA, our proposed work will exert a positive public health impact addressing a common and expensive public health problem.

NIH Research Projects · FY 2025 · 2022-09

Project Summary One of the main research directions of my laboratory focuses on regulation of the endothelial barrier and endothelial cell migration. These processes are critical for physiological function of vascular system and they are often dysregulated in human diseases. A lot of progress has been made in understanding signaling that regulates endothelial barrier and cell migration. However, stimulation of endothelial cell migration during angiogenesis is a highly localized and transient event. Defining the role of the local and temporal components of angiogenic signaling has been challenging due to limitations of current tools. Furthermore, spatiotemporal regulation of the endothelial barrier by these stimuli has been poorly understood. Our proposed work will focus on determining how the location and duration of migratory signals direct endothelial cell invasion and migration through extracellular matrix, and how they affect the organization and permeability of the endothelial barrier. The endothelial barrier is controlled at the level of adherens junctions (AJs), cell-cell adhesion structures mediated by the transmembrane protein VE-cadherin. Phosphorylation-mediated signaling regulates the structure and permeability of AJs. In our recent studies, we described a dual role of tyrosine kinase Src and its phosphorylation of VE-cadherin in regulation of endothelial permeability. Our results demonstrated that Src- mediated phosphorylation induces formation of dynamic AJs that still retain their barrier function. This suggests a mechanism for the regulation of AJ plasticity that does not compromise barrier permeability during endothelial cell migration. In parallel studies, we dissected a mechanism of Src-regulated degradation of the extracellular matrix by the endothelial cell and discovered a novel cytoskeletal component that mediates formation of matrix-degrading podosomes. The studies proposed here will continue to build on our previous findings and focus on dissecting how phosphorylation of VE-cadherin and angiogenic signaling by Vascular Growth Factor Receptor 2 (VEGFR2), Sphingosine-1-phosphate Receptor 1 (S1PR1), and Src regulate plasticity of AJs as well as invasion and migration of endothelial cells. We will employ novel optogenetic tools that will allow us to interrogate these processes with precise spatial and temporal control. We will use engineered light-regulated VEGFR2, S1PR1, and Src to determine the effects of locally and temporally controlled angiogenic signals and dissect mechanisms that mediate regulation of AJs and migration of endothelial cells in three dimensional environment. Our long-term goal is to define the processes that control migration of endothelial cells and endothelial barrier function during angiogenesis.

NIH Research Projects · FY 2025 · 2022-09

Substantive research training early during the course of medical school education is critical to address the declining number of physicians choosing research careers. The proposed University of Illinois Short-Term Research Training Program (SRTP) will provide first year medical students (12 slots in Year 1, 15 in Years 2-5) with a two month mentored research experience in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) mission areas including kidney, urologic and hematologic diseases; digestive disease and nutrition; and diabetes, endocrinology and metabolic diseases. The University of Illinois College of Medicine (UI COM) is the largest medical schools in the United States. The program will draw from first-year medical students at UI COM, as well as other medical schools in the Chicago metropolitan area and the Midwest. We have assembled an outstanding team of mentors in NIDDK mission areas with expertise in basic/mechanistic, clinical/translational, and behavioral/community-based research. In addition to mentored research, students will participate in an interactive, structured curriculum focused on research methodology, responsible conduct of research, and career development. To learn presentation skills and principles of rigor and reproducibility, students will co-lead, with a research mentor, journal clubs and works-in-progress sessions. Additional novel aspects of the program include the use of peer-to-peer mentoring and ongoing exposure to successful early career physician-scientists. At the end of the summer, students will present at the SRTP Research Symposium and the COM Research Day. After the program is completed, the program will utilize multiple strategies to facilitate continued involvement in research activities, including encouraging students to apply to the James Scholar Program, a program that provides the infrastructure to support research during the second to fourth years of medical school. In addition, the program will strive to create a sense of community among SRTP cohorts by hosting quarterly “Ideas on Tap” (research/social networking events at which students present ongoing research) and will maintain connections with trainees using the program website, newsletters, an SRTP Slack communication channel, and social media. An Executive Committee will oversee implementation of the program, recruitment/selection, program evaluation, and tracking of short- and long-term outcomes. Strengths of the program include the experienced co-Directors, committed and well-funded program mentors with broad expertise across NIDDK mission areas, an innovative structured curriculum, a robust recruitment plan, strong institutional support, and a thoughtfully conceived plan to foster continued research involvement after program completion. Consequently, the proposed SRTP will provide medical students with substantive research experience, facilitating the achievement of the long-term objective of increasing the pipeline of physicians entering the research workforce in NIDDK mission areas.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Both soft and hard tissue wound healing are impaired in type 2 diabetes (T2DM). Diabetes negatively impacts fracture healing, bone regeneration and osseointegration of endosseous implants. The complex physiological changes associated with diabetes are often manifest in immunological responses to wounding and repair where macrophages play a prominent role in determining outcomes. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and immunomodulation of bone repair is affected by key interaction involving macrophages and mesenchymal stem cells (MSCs). Yet, a fundamental knowledge gap exists with respect to the nature of and the mechanisms that govern this interaction in the presence of T2DM. My recent published study has revealed that the conditioned medium from diabetic mouse macrophages impairs osteogenic differentiation of MSCs. My studies also show that macrophages secrete phenotype-dependent extracellular vesicles (EVs) that affect the level of bone repair. Here, I hypothesize that diabetic macrophage EVs mediate specific paracrine control of osteogenesis. To test this hypothesis, I propose two independent but thematically related aims. In Aim 1, I will characterize and compare wild type mouse macrophage EVs (wtEVs) and diabetic mouse macrophage EVs (dbEVs) at the structural and functional level and define the effects of these EVs on MSC osteogenic differentiation quantitatively. I will demonstrate the role of macrophage miRNAs cargo on osteoinduction by interfering miRNA function in Argonaute 2 (involved in RISC complex formation and miRNA function) knockdown MSCs. In addition, I will validate that dbEVs contain miRNAs that negatively influence the process of osteogenesis by generating EVs from DICER (required for mature miRNA formation) knockout macrophages. In Aim 2, I will identify significantly distinct miRNAs in dbEVs, their target genes and signaling pathways involved in osteogenesis by bioinformatic approach. I will evaluate the functionality of these miRNAs on osteoinduction by utilizing mimics/antagomirs that increase or eliminate the effects of identified miRNAs. I will study selected target genes at the level of miRNA interaction to affirm the direct effects on gene regulation and downstream effects of these key miRNAs on osteoinductive pathways. Translationally, I will generate functionally engineered EVs (FEEs) by engineer the candidate miRNAs (promote osteogenesis) that rescue dbEV effects into MSC EVs. The FEEs will be characterized structurally and functionally in vitro. Further I will utilize calvarial bone defect model to evaluate the function of selected miRNAs within FEEs on bone healing in diabetic mice. Overall, these mechanistic studies will explore the significance of the macrophage EV-mediated immunomodulation that occurs between macrophages and MSCs in the context of bone healing in the presence of T2DM. These studies will refine knowledge of diabetic pathology and provide potential targets for therapeutic intervention.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY Nearly 94% of breast cancer survivors experience one or more symptoms during or after endocrine therapy. Joint pain, hot flashes, sleep disturbance, fatigue, depression, and anxiety are the most common concurrent symptoms, some of which can persist for 5 to 10 years. These symptoms negatively affect adherence to treatment and compromise people’s functional status, quality of life, and work functioning. Acupuncture is a single therapy with few side effects that holistically addresses multiple symptoms. The effect of acupuncture on multiple symptoms among breast cancer survivors receiving endocrine therapy has not been investigated. Further, implementation-focused acupuncture research focuses on privately insured clinic settings and ignores social determinants of health. There is an urgent need to remove barriers and ensure equal access to this evidence-based treatment among breast cancer survivors with limited access to acupuncture. Federally qualified health centers (FQHC) provide care to people who experience significant barriers to health care access. To date, limited data exist about the use of acupuncture among cancer survivors attending FQHCs. This research study has 2 aims: (1) to test the feasibility and acceptability of implementing acupuncture within an FQHC oncology clinic as a way to manage multiple symptoms (pain, hot flashes, fatigue, sleep disturbance, depression, anxiety) among breast cancer survivors receiving endocrine therapy, and (2) to use a mixed methods approach to identify barriers and facilitators associated with the implementation of acupuncture within an FQHC. The long-term goal is to facilitate the widespread implementation, dissemination, and sustained utilization of acupuncture for symptom management among medically underserved breast cancer survivors receiving endocrine therapy in FQHCs nationwide, and ultimately to promote broader insurance coverage for acupuncture. The proposed research is significant because of its potential to ensure equal access acupuncture, an evidence-based intervention. Results will provide the foundation for a larger multi-site hybrid effectiveness-implementation trial of integrating acupuncture into services provided at FQHCs for breast cancer survivors.

NIH Research Projects · FY 2026 · 2022-08

Abstract Despite much progress, alcoholic liver disease (ALD) remains a major health problem worldwide. The disease process is characterized by early steatosis, steatohepatitis, with some individuals ultimately progressing to fibrosis/cirrhosis and liver failure. Unfortunately, there is currently no accepted therapies available to halt or reverse this process in humans. Nicotinamide N-methyltransferase (NNMT) catalyzes SAM-dependent degradation of nicotinamide, a predominant precursor for cellular NAD+ biosynthesis via a salvage pathway. SAM (s-adenosylmethionine) is the first product of methionine metabolism and a universal methyl donor in cellular transmethylation reactions. The critical role of NNNT in regulating both NAD+ and SAM homeostasis make it an emerging novel metabolic regulator. We are the first to report that the liver ATF4 transactivation plays a mechanistic role in mediating NNMT upregulation in the setting of chronic alcohol consumption and adenoviral shRNA knockdown of NNMT is protective against alcoholic fatty liver development, suggesting that NNMT can be an ideal therapeutic choice for ALD treatment. The preliminary data recently obtained from our laboratory uncovered that NNMT inhibition was associated with improved mitochondrial unfolded protein response (UPRmt) and blunted hepatic PPAR-gamma activation upon chronic alcohol exposure. In this proposal, we will further elucidate the mechanistic implication of NNMT in the pathogenesis of ALD. Successful performance of the studies proposed in this proposal will not only shed new light on the pathogenesis of this disease, but also pave the way for novel therapeutic interventions for ALD. The three aims are included in this proposal to test our hypothesis: AIM 1: To delineate mechanism(s) underlying NNMT-associated liver pathologies in ALD. Both animal and cell culture studies will be conducted to elucidate mechanism by which NNMT inhibition improves UPRmt elicitation in the liver and to determine the mechanism whereby NNMT upregulation contributes alcohol-induced liver PPAR-gamma activation. AIM 2: To elucidate mechanism(s) by which chronic alcohol consumption leads to hepatic ATF4 activation and NNMT upregulation. Both hepatocyte- specific Gcn2 knockout mice and hepatocyte-specific arginase-1 overexpressing mice will be fed with isocaloric control or alcohol-diet for 5 weeks. Targeted metabolomics will be conducted to using primary hepatocytes to quantify the effects of alcohol on hepatic arginine metabolism. AIM 3: To determine both preventive (the pathogenic role) and therapeutic potential of NNMT-targeting approach for ALD. Animals with liver-specific NNMT knockout will be exposed to either isocaloric control or alcohol-diet. Both preventive and therapeutic efficacy of NNMT inhibition for ALD will be evaluated.

NIH Research Projects · FY 2024 · 2022-08

Getting to Zero (GTZ) new HIV infections by 2030 is a public health priority. Increasing HIV prevention, self- management, and harm reduction among at-risk populations is critical. HIV prevalence in sex workers is 12 times greater than the general population, highlighting the need for targeted prevention efforts. Increasing optimal use of pre-exposure prophylaxis (PrEP) in this population will advance progress toward GTZ 2030. Community empowerment interventions reduce HIV risk among sex workers because interventions are collaboratively designed, implemented, and evaluated by the target population. A community-empowered PrEP navigation approach may be feasible and acceptable among sex workers. One such approach is a group healthcare model called Centering Healthcare (Centering), which originated as a group prenatal care model and has since been modified for various patient populations. Centering's three core components: healthcare, interactive learning, and community building, disrupt typical healthcare power hierarchies by creating a respectful and collaborative interactive learning environment that honors group needs. The model's positive impact links self-management goals and health assessment with interactive activities to foster health promotion. Using an implementation science framework and building on Centering intervention effectiveness, this study will be the first to evaluate whether Centering is feasible and acceptable for PrEP education, navigation, and adherence among sex workers. Preliminary qualitative research with 39 sex workers in Chicago highlighted that stigma and healthcare discrimination dissuaded sex workers from regularly accessing HIV prevention information, services, and treatment. In collaboration with sex workers and a Community Advisory Board (CAB), we culturally adapted the Centering curriculum and facilitator's guide to meet the stated needs of sex workers. Guided by an Exploration, Preparation, Implementation, Sustainment framework (EPIS), the overall goal of this study is to expand on my formative research and evaluate if this culturally adapted Centering PrEP (C-PrEP+) model is feasible and acceptable for addressing education, navigation and PrEP adherence needs of HIV negative sex workers in Chicago. Specific aims of this study are to 1. Produce an implementation plan for integrating C-PrEP+ into a Federally Qualified Health Care (FQHC) system. 2. Conduct a pilot feasibility and acceptability trial of C-PrEP+. 3. Complete an evaluation of C-PrEP+ to document implementation barriers and facilitators. The research plan will be augmented by expert mentoring and didactic research training at University of Illinois Chicago and University of California San Francisco. This proposal, mentoring, and coursework will provide essential career development in: 1. Quantitative methods, 2. Health intervention study design, preparation, implementation, and 3. Mixed-methodological evaluation. This foundation will uniquely position the PI to lead a fully powered multi-site RCT to determine if C-PrEP+ is an efficacious and sustainable model to address HIV prevention among sex workers.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Cytokines represent a broad range of small signaling proteins utilized by immune cells in cell-to-cell communications. Dysregulated cytokine release contributes to acute and long-term conditions, from life- threatening cytokine release syndrome to delayed wound healing. Targeting the cytokine release is an attractive strategy because it can regulate multiple cytokines involved in complex diseases. However, no FDA- approved drugs target this process, and related clinical trials remain scarce. The lack of clinical translation represents an urgent need to develop advanced technologies to better understand the cytokine release process at the molecular level. To date, existing assays for cytokine quantification, such as ELISA and flow cytometry, provide a low resolution that masks detailed mechanistic information in space and time. By capitalizing on the PI’s unique expertise in immune imaging, the R35 proposal will address this need by developing ultrasensitive cytokine quantification techniques using T cells and interleukin-2 as a model system. Cytokine quantification will be achieved in three specific areas: 1) at the resolution of single-vesicle fusion events with the plasma membrane, 2) at the nanoscale membrane release sites, and 3) in the membrane- enclosed form of extracellular vesicles (EVs). Enhanced mechanistic understanding will be obtained at the single-cell level regarding the temporal and directional profiles of cytokine release, proximity-based regulation by membrane calcium channels, and the dynamic distribution between soluble and EV-associated cytokines during T cell activation. Each of these areas will potentially enable compound screening targeting specific spatial and temporal phenotypes, investigations of membrane channel inhibitors, and targeting EVs for cytokine modulation. Future studies will be expanded to other essential cytokines from adaptive and innate immune cells. Ultrasensitive quantification of cytokines will enhance mechanistic understanding of the cytokine release in search of novel membrane targets to modulate the process.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Human induced pluripotent stem cells (iPSCs) can be used to generate 3-dimensional lung organoid structures. However, most lung organoid studies have focused on human iPSC-derived lung epithelial subtypes. They have not to date included human iPSC-derived endothelial cells and systematically addressed the critical role of lung vascular endothelial cells and vascular perfusion itself in the generation and maturation of lung organoids which model human lung structures. The alveolar units consist of two predominant cell types – epithelial cells (EpiC)(40-45% of total cells) and endothelial cells (EC) (45-50% of total cells). Our key Supporting Data support the critical and heretofore underestimated role of human lung vascular endothelial cells in guiding differentiation of human lung epithelial progenitor cells and formation of vascularized human lung organoid. We propose to use this novel platform generated by integration of hiPSC-derived epithelial and endothelial cells to address the following aims: Aim 1 tests the hypothesis that endothelial cell-derived angiocrine signals in lung organoids activate Wnt signaling and mediate the maturation of lung alveolar units and the corollary hypothesis that reciprocal epicrine signaling of EpiC regulates lung EC fate, generation of recently described specific lung EC populations and lung microvessel patterning at the level of alveoli. Aim 2 will test the hypothesis that the vascularized and perfused human lung organoid serves as a translationally relevant reductionist model for teasing apart the elusive signaling and molecular mechanisms of inflammatory injury at the level of the alveolar unit and resolution of injury. Aim 3 will test the hypothesis that lung EC signaling through the upregulation of ACE2 in alveolar Type II epithelial cells promotes SARS-CoV-2 entry and infection of lungs. Together the proposed studies through their focus on lung EC and vascularization of human lung organoid and incorporation of alveolar epithelial cells in this system will uncover fundamental mechanisms of how the vascularized alveolar unit functions in health to maintain homeostasis and how defective cross-talk between EC and alveolar epithelial cells contributes to inflammatory lung disease.

NIH Research Projects · FY 2025 · 2022-08

TITLE Pyroptosis is a Programmed Trial-by-Fire. PROJECT ABSTRACT The inflammatory cell death program pyroptosis occurs in a wide range of cell types and plays important roles in development, tissue injury, and tumor growth. Curiously, unlike other cell death mechanisms, cells frequently survive pyroptosis activation. The persistence of a family of “inefficient” cell death genes is puzzling and suggests they may have other roles. Intuitively, cells that survive the inflammatory insult will dictate both the short-term adaptation and long-term repair and regeneration afterwards. Therefore, a flawed death program may actually be a “trial”. However, the details of cell survival after pyroptosis and the biochemical circuitry beyond its activation are under-studied, hindering our basic understanding. In this Program, we hypothesize that cell death is a side- effect of pyroptosis, which in fact aims to reprogram cell functions. To explore the new concept that “trial-by-fire” provides strong evolutionary advantages, we will investigate 1) whether it is possible to stop/resume pyroptosis and the basic mechanisms used to exert such control; 2) the capacity and limits of pyroptosis signaling; and 3) the single cell phenotypic effects of weathering pyroptosis. In pursuing this central hypothesis, we will discover new cell signaling circuits and simultaneously enhance the field’s ability to detect novel pathways by developing new genetically encoded tools, imaging algorithms, and bioinformatics methods. The Program aims to construct a new perspective that the salient function of pyroptosis is to prime and educate through a near-death experience.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT The discovery of new small molecules that perturb the function of membrane receptors, like G-protein coupled receptors (GPCRs) and ligand-gated ion channels (LGIC), remains critically important to the study and improvement of human health. Natural products are particularly well-suited for this task, as their structural complexity and unique mechanism of action make them superior chemical probes and excellent starting points for drug discovery. The Riley lab is focused on developing step-economic synthetic routes and robust isolation protocols to access these complex natural product scaffolds. Through modular total syntheses, semi-synthetic methods, and contemporary receptor assays, we transform natural products into highly potent and selective tools for studying membrane receptors. This application describes an overview of our work and future directions in applying these strategies to investigate the nicotinic acetylcholine receptors (nAChRs) and the kappa opioid receptor (κOR) as representative LGIC and GPCR, respectively. The first research area builds upon our work that recently identified members of the Aristotelia alkaloid family as potent inhibitors of the nAChRs, a major class of LGICs, with an unusual yet desirable subtype-selectivity. During this award, we will develop streamlined synthetic chemistry to access the entire class of Aristotelia alkaloids and generate large libraries of their derivatives. By coupling these synthetic chemistry efforts with an expanded ability to screen for activity against an array of nAChR subtypes and other membrane receptors, this work will deliver new chemical tools to probe the biological function of specific nAChR subtypes. In the second research area, we will explore a novel class of κOR agonists derived from the indole alkaloid akuammicine that were recently discovered in our laboratory. Our initial studies identified these akuammicine derivatives are potent biased agonist that preferentially activate the G-protein signaling pathway. Leveraging isolation protocols that provide synthetically useful quantities of complex alkaloids directly from their natural sources, we will employ late-state diversification techniques to rapidly generate novel derivatives that probe ligand-receptor interactions within the κOR and monitor their ability to initiate opioid signaling cascades. We expect the results from this research will reach beyond the nAChR and κOR and can be applied to other therapeutically relevant LGICs and GPCRs, thereby having a significant impact on drug discovery by revealing new directions to rationally design ligands for these important membrane receptors.

NIH Research Projects · FY 2025 · 2022-07

Rheumatoid arthritis (RA) is the most common autoimmune disease, which affects 2.5 million people in the US. It has been shown that joint monocyte infiltration and differentiation into inflammatory macrophages (MΦs) play a key role in disease progression. Patients with a positive response to RA therapy, exhibit a reduced number of MΦs, joint inflammation, pain & radiological damage. In contrast, in non-responsive patients, the number of inflammatory MΦs is expanded along with skewed metabolic rewiring towards glycolysis and away from mitochondrial oxidative phosphorylation. Hence, to find a novel therapeutic strategy, there is a critical unmet need to elucidate the molecular mechanism by which naïve joint cells are reprogrammed into glycolytic RA MΦs. We show for the first time, that a specific cytokine rewires the naïve M0 cells into glycolytic MΦs that produce high levels of inflammatory monokines and metabolites. Notably, these glycolytic MΦs are primed to differentiate into mature osteoclasts. Notably, dysregulation of syndecan (SDC)1 impairs secretion of the inflammatory monokines, polarization of the glycolytic CD14+CD86+GLUT1+ MΦs, and remodeling of the primed glycolytic cells into mature osteoclasts facilitated by the cytokine of interest. Based on these novel observations, we hypothesize that binding of the cytokine of interest to SDC1 reprograms the naïve cells into glycolytic MΦs and mature osteoclasts, and blockade of SDC1 or the activated metabolic intermediates will attenuate arthritis. To test our hypothesis, we will determine if inhibition of the SDC1 or the identified glycolytic intermediates will impede the remodeling of naïve cells into metabolically active RA MΦs and mature osteoclasts using the early and late-stage patients. Next, we will delineate if the adoptive transfer of fully differentiated glycolytic MΦs can restore preclinical arthritis in the absence of metabolic factors linked to SDC1. Last, we will investigate whether deregulation of SDC1 or the master regulator of glucose metabolism will attenuate experimental arthritis. By integrating mechanistic RA cellular studies and preclinical models, we aim to delineate pathways by which the metabolically active MΦs advance joint disease. Our proposed approach will answer several fundamental questions including; 1) What are the metabolic machinery activated in RA MΦs and experimental arthritis, 2) Whether RA MΦ-regulated inflammatory and erosive phenotypes will be reversed by dysregulation of glycolytic intermediates and 3) Does targeting the hypermetabolic activity in MΦs represents a new therapeutic approach for RA.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT The 2017 publication of a landmark New England Journal of Medicine study linking clonal hematopoiesis of indeterminate potential (CHIP) to atherosclerotic cardiovascular disease (ASCVD) ushered in a new paradigm in vascular aging research. This work highlighted age-related somatic DNA mutation as an important contributor to cardiovascular health, with CHIP thought to promote ASCVD primarily through inflammatory sequelae. Despite the pro-inflammatory setting of chronic kidney disease (CKD), CHIP has not been assessed for its contribution to end stage kidney disease and premature ASCVD among these patients. Hence, our long-term objective is to characterize the role of CHIP and its related mechanisms in incident CKD progression and ASCVD in a CKD setting. To achieve this overall objective, we will leverage the rich resources of the Chronic Renal Insufficiency Cohort (CRIC) study, utilizing available whole exome sequencing (WES) data for baseline CHIP measurement, longitudinally ascertained biospecimens, clinical information, and molecular biomarkers, along with CKD progression and ASCVD events collected over 16 years of follow-up study. We propose a discovery stage cohort of 2,126 CRIC participants who were 65 years of age or older at baseline. Older adults are selected to maximize study efficiency, since CHIP is rare in younger adults. CHIP status will be determined using baseline WES data and our state-of-the-art analytic pipeline for CHIP somatic variant calling. We also propose repeated CHIP measurements at 3- and 6-years follow-up in the entire older adult sub-cohort, along with RNA sequencing (RNA-seq) in 400 CRIC participants, half with CHIP. We will test CHIP associations with incident CKD progression (Aim 1) and ASCVD (Aim 2) among the 2,126 CRIC CKD patients. We will then evaluate upstream (Aim 3a) and downstream (Aim 3b) CHIP mechanisms in the unique CKD setting using longitudinal information on CHIP and known risk factors for CKD progression and ASCVD, including: clinical variables (blood pressure, glycemic traits, and lipids), biomarkers of kidney injury, cardiac stress and injury, inflammation, and fibrosis, use of renin angiotensin aldosterone system inhibitors, and aging related genetic factors (CDKN2A variants). To discover novel molecular mechanisms, our RNA-seq study will test associations between CHIP and gene expression (Aim 4a). Differentially expressed genes will be evaluated for association with CHIP mechanisms identified in Aims 3a and 3b (Aim 4b). To replicate findings, we will leverage existing CHIP, clinical, biomarker, gene expression and outcome data in up to 4,126 Trans- omics for Precision Medicine program and 8,520 UK Biobank participants. CHIP effects will be precisely estimated in powerful meta-analyses of discovery and replication studies. Findings of the proposed research could have broad implications, ranging from the improvement of risk stratification efforts to the development of personalized strategies and novel molecular-based therapies for ESKD and ASCVD prevention in CKD.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Niemann-Pick disease, type C1 (NPC1), is an autosomal recessive, neurovisceral disorder, and patients typically succumb to complications of the disease in the early adulthood years. The clinical phenotype of NPC1 is broad including both central nervous system and peripheral dysfunction and currently there is no FDA- approved therapy. The enclosed proposal seeks to develop and understand the mechanism of action of a new class of peptides to ameliorate cholesterol storage and associated phenotypes of NPC1. Our central approach is to address the biochemical deficits upstream of the NPC1 protein. First, we will understand how defined protein and lipid biomarkers respond to peptide treatment. Second, we will perform a DMPK study and investigate lifespan extension with treatment. Third, we will investigate the mechanism of action by which these peptides reduce cholesterol storage. To carry out the proposed project, we will leverage our expertise in mass spectrometry, biochemistry and molecular biology techniques.

NIH Research Projects · FY 2025 · 2022-07

Project summary The extracellular matrix (ECM) is a complex meshwork of hundreds of proteins that constitute the scaffold that holds our cells together. However, the functions of the ECM extend far beyond its structural roles. ECM proteins provide biochemical signals, either directly, by binding to cell surface receptors, or indirectly, by modulating growth factor signaling, that regulate many essential pathways controlling cellular functions, from proliferation and survival to migration and differentiation, all key to tissue and organ functions. Alteration of the ECM is linked to many diseases, including congenital diseases (e.g., Marfan syndrome, Alport syndrome, Ehlers–Danlos syndrome), musculo-skeletal diseases (e.g., osteoarthritis, myopathies), cardiovascular diseases, fibrosis, and cancer. Yet, despite its importance, the ECM remains largely underexplored. For example, we have yet to decipher the ECM protein composition (or “matrisome”) of organs, of tissues, and, within tissues, of specialized niches. We also do not fully understand which cell types produced which ECM proteins, nor do we know how the composition of the ECM changes over time and during diseases. These gaps in knowledge are mainly due to the lack of adequate methods to study the ECM. The secretion and post-translational modifications that accumulate in the ECM over time are critical for proper ECM functions and cannot be fully studied by RNA-level observations only. Thus, protein-level evidence is key to understand the function and dynamics of the ECM. However, ECM proteins, being typically very large, heavily post-translationally modified, and, overall, highly insoluble, are under-represented in global proteomic datasets. We propose to fill these gaps in knowledge by contributing our expertise in ECM biology, ECM proteomics, and computational biology to the technology- development and mapping efforts of the Human BioMolecular Atlas Program (HuBMAP), and ultimately build spatially-resolved maps of the matrisome of all organs. To achieve this goal, we will pursue the following aims: 1) re-analyze the vast amount of single-cell RNA-seq data generated by HuBMAP to identify the cell populations expressing ECM and ECM receptor gene transcripts for all organs, 2) integrate existing imaging data and mass spectrometry data generated by the HuBMAP to build a model to predict protein co-expression and create spatially-resolved tissue maps of the ECM, 3) contribute our 10+ years of expertise in ECM proteomics to ensure the effectiveness of future data collection, to capture ECM-relevant information, by members of the HuBMAP. For our efforts to benefit the entire scientific community, we will deploy all datasets and technologies via the HuBMAP portal and via MatrisomeDB, the ECM protein knowledge database we have previously developed. This mapping effort will constitute a first step toward understanding the roles of the ECM in health and diseases and toward the development of future ECM-focused diagnostic and therapeutic strategies.

NIH Research Projects · FY 2025 · 2022-07

Abstract Macrophage Immunosuppression by Quorum-Induced Streptococcus pyogenes The human-restricted pathosymbiont Streptococcus pyogenes (Group A Streptococcus, GAS) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, allowing coordination of biofilm formation and lysozyme resistance. Preliminary findings demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. Published and preliminary data show that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NFB activity, was substantially suppressed upon interaction with QS-ON GAS but not QS-OFF bacteria. Neither macrophage viability nor bacterial adherence were seen as different between QS activity states, yet TNF, IL-6, INF levels and an NFB reporter were drastically lower when QS was ON. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Newly acquired transcriptomic analysis (RNA-Seq) of macrophages infected with QS-ON and QS-OFF GAS indicate clear divergence in gene expression patterns between infection types. QS-OFF infections induce macrophage characteristics with signatures of classic activation (M1-like), whereas QS-ON infections produced genetic signatures consistent with alternatively activated (M2-like) macrophages, where metabolic pathways of oxidative phosphorylation and fatty acid beta-oxidation are induced. We propose a model that upon contact with macrophages, QS-ON GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-ON GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. This application seeks to accomplish three primary objectives: 1) identify the QS-regulated factor generated by the BGC and the biosynthetic intermediates; 2) identify the macrophage target and mechanism of NFB inhibition; and 3) evaluate the physiological impact on immune cell activity and the advantage provided to GAS in vivo and in human explant tissue models.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Listeria monocytogenes (Lm) is a gram-positive food-borne intracellular bacterial pathogen that is capable of causing serious invasive disease in humans. As a widespread environmental organism, Lm is a frequent contaminant of food processing facilities and has been responsible for some of the largest, most expensive, and most deadly food recalls in US history. Lm is one of a select number of pathogens that is transmitted during pregnancy from mother to fetus. These infections can be devastating, as often an infected woman does not even realize she has been infected with Lm until she miscarries or gives birth to a stillborn or systemically infected infant. The high mortality rate and devastating sequelae that accompany Lm invasive disease despite antibiotic treatment underlie the critical need for new therapeutic strategies to safely and effectively manage Lm invasive infections. We have recently discovered that select isolates of Lm have an enhanced ability to target the placenta and fetus based on increased expression of the bacterial surface protein InlB. Increased InlB is sufficient to transform a strain that normally exhibits a low frequency of fetal colonization to a strain that is capable off nearly 100% fetal infection. Naturally occurring amino acid variations within InlB may both increase protein stability and enhance stimulation of c-Met, the host growth factor receptor bound by InlB. Met is abundantly expressed by placental tissue and is required for embryonic and placental development. We hypothesize that Lm strains expressing select variants of InlB exhibit enhanced invasion through the manipulation of c-Met signaling pathways, leading to increased rates of fetal transmission. These strains additionally stimulate a robust immune response that leads to placental barrier dysfunction and fetal death. The specific aims of this proposal will undertake a functional assessment of Lm InlB to reveal molecular mechanisms underlying vertical transmission as well as examine the contributions of maternal and fetal immune signaling to pregnancy outcome. Aim 1 will functionally define the mechanisms underlying InlB surface localization and activity. This aim will define mechanisms that contribute to InlB stability at the bacterial cell surface and will examine functional differences between surface localization and secretion. Aim 2 will decipher the mechanisms underlying InlB enhancement of Lm vertical transmission. We will examine and compare portals of Lm entry in pregnant mice, and explore host responses to Lm infection that influence pregnancy outcome. Aim 3 will explore maternal and fetal defenses triggered by high efficiency vertically transmitted strains that contribute to pathology. These studies will clarify how select Lm isolates gain access with high efficiency to placental/fetal tissues to cause devastating forms of neonatal disease and death.