Case Western Reserve University

NIH Research Projects · FY 2025 · 2021-09

Project Summary - Overview The newly developed CWRU (Case Western Reserve University) Center for Excellence on the Impact of Substance Use on HIV was conceived in August 2017 and was initiated to effectively promote excellence in basic, translational, and clinical research concerning the intersection between substance use and HIV. The Center for Excellence includes 12 NIDA-funded Investigators who collectively hold 9 R01, 2 DP1, 3 R34, 2 R44, 1 U01, and 2 K01 drawn from the CWRU School of Medicine, University Hospitals Cleveland Medical Center, MetroHealth Medical Center, the Cleveland Clinic Foundation, the Louis Stokes Cleveland Veterans Administration Medical Center, the CWRU Mandel School of Applied Social Sciences, the Begun Center for Violence Prevention Research and Education, and the Cuyahoga County, Lorain County, and City of Cleveland Departments of Health. Major research strengths in the Center for Excellence include impact of drug use on HIV immunopathogenesis, HIV latency and Cure, NeuroAIDS, gastrointestinal dysfunction, sexual risk behavior, and HIV and HCV co-infections. Our studies are anchored by advanced computational, systems biology, biomimetic models, and induced pluripotent stem cell (iPSC) technologies that provide the opportunity to perform meta-omics analyses of the impact of opioid, methamphetamine, cannabis, and cocaine misuse on several clinical, virological, immunological, behavioral, and neurological outcomes of HIV disease. The Center for Excellence has the following Specific Aims: • Provide scientific leadership to position the Center at the forefront of substance use disorder research and function as a national research resource for the study of drug use in persons with HIV. • Establish an administrative infrastructure that maintains fiscal and management oversight of the Cores. • Apply advanced Computational Biology, Primary Cells, Biomimetic models, and iPSC- derived Cells to study of the impact of substance use on HIV disease. • Support translational, clinical, and behavioral research through access to unique clinical cohorts of persons with substance use disorder with HIV and at risk for HIV. • Accelerate junior faculty development and encourage experienced faculty to enter the substance use research arena to support the next generation of investigators. • Participate in community outreach.

NIH Research Projects · FY 2025 · 2021-09

Although significant progress has been made in understanding the genetic origins of neurodevelopmental disorders, it remains unclear what specific molecular steps are disrupted and in which specific neurons types they are dysfunctional or inoperable. One potential reason for this lack of understanding is that neurons, originally classified according the type of transmitter produced, are now well known to possess substantial molecular, cellular, and functional heterogeneity. It seems plausible that neurodevelopmental disorders may arise not only from developmentally altered identities of an entire population of one particular neuron type but also from altered development of one of its specific molecular or functional subtype(s). There has been a decades-long intense interest in the regulatory mechanisms controlling 5-HT neurons as 5-HT has wide-ranging modulatory effects on central neural circuitry and dysfunction of the serotonergic system has been implicated in several neuropsychiatric diseases including depression, stress-related anxiety disorders, autism, intellectual disability, OCD, and schizophrenia. 5-HT neurons possess tremendous molecular, morphological, and electrophysiological heterogeneity. However, developmental trajectories of 5-HT neuron subtypes are currently unknown as are the regulatory mechanisms that govern their development. The objective of the proposed research is to use single cell RNA-seq and single cell ATAC-seq to comprehensively define the spatiotemporal developmental trajectories of 5-HT neuron subtype transcriptomes and chromatin accessibility. We will combine recent advances in single-cell genomics methods together with our well- established serotonergic transgenic tools, our extensive experience in flow sorting mouse 5-HT neurons, and our bioinformatics expertise to investigate the development of single-cell 5-HT neuron transcriptomes and chromatin accessibility throughout fetal to early postnatal maturation. We will also investigate at the single cell level, the hypothesis that the two disease-associated terminal selectors in 5-HT neurons, Pet1 and Lmx1b, function to determine postmitotic 5-HT neuron subtypes through differential regulation of subtype-specific gene expression, subtype-specific chromatin accessibility and control of downstream subtype-specific transcription factor codes. Pet1 is of special interest as homozygous knockout mutations in FEV, the human ortholog of Pet1, were recently reported in two brothers with Intellectual Disability and Autism Spectrum Disorder. In Aim 1, we will define the developmental trajectories of 5-HT neuron subtypes. In Aim 2, we will investigate the control of 5-HT neuron subtype transcriptomes by Pet1 and Lmx1b. In Aim 3, we will determine the chromatin mechanisms involved in the generation of 5-HT neuron subtypes. The completion of our proposed aims will lead to a greater understanding of the subtype-specific gene regulatory networks that generate 5-HT neuron subtypes, which may help illuminate specific neurogenetic pathways that are disrupted in neurodevelopmental disorders such as ASD/ID.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The long-term goal of this project is to determine the clinical impact of metastasis-directed radiotherapy (MDT) in men with de novo oligometastatic prostate cancer (PCa), and identify which men may be cured and benefit most from MDT. We aim to achieve this goal through the conduct of a phase 3 randomized controlled trial with prospective imaging and biospecimen (e.g. tissue and blood) collection. This trial is novel in that it is a randomized North American sub-study (n=200) of the next arm (Arm M) of the international landmark multi- arm, multi-stage, STAMPEDE trial. The ability to conduct this trial with comprehensive biospecimen collection and imaging analysis will be achieved through our unique research team across extramural and intramural centers, comprised of experts in prognostic and predictive biomarker signature identification, bioinformatics, biostatistics, genomics, imaging, and clinical trial execution. We will leverage the opportunity for North America to participate in the STAMPEDE trial to not simply identify the true impact of MDT in the first ever large phase 3 trial testing MDT in PCa, but use this one of a kind clinical trial working with the NCI to develop the first predictive biomarkers of benefit of MDT using radiomics of conventional and molecular PET imaging, as well as sequencing of primary, metastatic, and liquid biopsies. This goal will be carried out through three specific aims. Aim 1 will focus on the conduct of the phase 3 randomized North American sub-study to determine if the addition of MDT to standard systemic therapy and treatment of the primary improves failure-free survival. Five centers will participate, including the NIH Clinical Center. These patients will all be included in the international STAMPEDE trial with the primary endpoint of overall survival. Aim 2 will leverage the baseline CT and bone scans collected on all patients, as well as a subset that will be sent to the NCI to have pre-treatment 18F- DCFPyL PET/CT scans performed (n=50). Radiomic analyses and image feature extraction will be performed, and this information will be used to identify which men benefit most from MDT. We hypothesize that a subset of men will benefit most from MDT and be identifiable through an imaging biomarker. Aim 3 will utilize the baseline prostate biopsy, metastatic biopsies, radical prostatectomy specimens, and liquid biopsies (circulating tumor cells and cell-free DNA), to annotate the molecular landscape of oligometastatic PCa. This data will then be used to develop a predictive biomarker to identify which men benefit most from MDT. International patient samples will be banked for later validation. We hypothesize that a discrete molecular profile will characterize which men are most likely to be cured from MDT. The impact of this work is extremely large, as it has the potential to cure a currently incurable subset of men with metastatic PCa. Successful completion of these aims would result in predictive biomarkers that could directly impact the clinical management of men with oligometastatic PCa, and transform current treatment paradigms.

NIH Research Projects · FY 2025 · 2021-09

This proposal describes a five-year mentored research and training plan that will facilitate the development of Dr. Stephanie Ford, MD as an independent investigator in the pathogenesis of congenital heart disease. Building upon Dr. Ford’s background as a clinical neonatologist and a basic scientist, she will attain expertise in design of mouse studies, RNA-FISH, and epigenetic mechanisms. She will gain skills through structured mentorship, hands-on laboratory experiences, didactic teaching, and formal classwork at Case Western Reserve University, FAES at the NIH, and Jackson Laboratories. Dr. Michael Jenkins, a pioneer in cardiac optical imaging, and Dr. Cynthia Bearer, an expert in prenatal alcohol exposure models, will provide their expertise and mentorship skills to this project, fostering Dr. Ford’s transition to research independence. An estimated 2.4-4.8% of newborns in the U.S. have fetal alcohol spectrum disorders (FASDs), caused by prenatal alcohol exposure (PAE). PAE induced Congenital Heart Diseases (CHDs) have not been studied as intensively as other FASD outcomes despite their high prevalence rate (40%). The CHDs associated with FASDs, mostly valvuloseptal and outflow tract defects, are life-threatening and impact growth and health. PAE is known to affect methylation and one-carbon metabolism. Normal one-carbon metabolism and its resulting methylation of DNA is crucial for the correct expression of genes. Comparative genomics studies have revealed that there is strong epigenetic conservation across vertebrate species including mice and avians, particularly the hyper-and hypo-methylated DNA sequences of critical genes. We will investigate the PAE-induced changes at times critical to heart development (endocardial cushion and 4 chamber development) in mouse and avian embryonic hearts. All hearts will be imaged with optical coherence tomography to rapidly determine their phenotype. DNA methylation changes will be determined with a combination of methyl-ATAC-seqand bisulfite sequencing. DNA methylation will be compared in both species, as conserved changes in two species are more likely to be relevant to human PAE-induced defects. RNA-FISH will be used to confirm gene expression changes, which will allow us to pinpoint where within the 3D heart, such as the forming valves, gene expression is changing. We will then explore the use of choline and glutathione to prevent the effects of PAE. Choline and glutathione are known to promote methylation in one-carbon metabolism. Choline has been shown in human studies to prevent early neurologic effects of PAE. We have shown in an avian model that glutathione prevents the CHDs and abnormal DNA methylation seen after PAE. We will use both avian and mouse models to determine the effects of alcohol + choline or glutathione on cardiac structure, DNA methylation, and gene expression. We hypothesize that by maintaining normal methylation, and therefore DNA expression, our chosen compounds will prevent the CHDs that result from PAE. Compounds that could prevent PAE-induced CHDs could help thousands of children and their families each year.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT (DESCRIPTION) The Case Western Reserve University (CWRU) Alzheimer’s Disease Translational Data Science (ADTDS) Training Program is designed to power therapeutic discovery by producing a new generation of scientists cross- trained in clinical research of Alzheimer’s Disease (AD) and analysis of large scale ‘omics data. Focused on predoctoral PhD and MD PhD students, this training program fills a critical gap between these disciplines in academic graduate programs. The ADTDS builds upon the Cleveland Alzheimer’s Disease Research Center (CADRC), and multiple ongoing national collaborations responsible for generating large-scale multi-omic data on diverse populations with AD. As part of this training program, trainees will: 1) Form multi-disciplinary mentorship teams to expand understanding of AD etiology and disease course through data analysis, 2) Interact with a variety of faculty who are world-recognized leaders in AD genetics, pathology, and neuroscience, 3) Access large-scale data from national initiatives supported by NIA, including the ADSP, ADGC, ADNI, and AMP- AD, to answer translational and basic research questions, and 4) Receive specialized training in responsible conduct and research reproducibility with special considerations for data science. Our cross-disciplinary training program provides strong mentoring and uses state-of-the-art data science experiences within the rich environment of the CWRU School of Medicine and its affiliated hospital systems to develop independent investigators who will lead the next generation of translational research in Alzheimer’s Disease.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Uterovaginal prolapse (UVP) is one of the most common conditions affecting women, with a 20% lifetime risk of UVP corrective surgery. Surgeries performed for UVP include either a vaginal or abdominal approach, with or without use of mesh, to correct defects in pelvic support. Accumulated evidence has shown that for post- hysterectomy pelvic organ prolapse (POP) repair, the use of mesh yields superior patient outcomes compared to vaginal repair without compromising patient safety. However, no high-quality data exists to help guide patients and surgeons on the best option for treatment of UVP of the two most commonly performed procedures: 1) vaginal hysterectomy with uterosacral ligament suspension (TVH+USLS) and 2) minimally invasive hysterectomy with sacrocolpopexy (MI-SCH+SCP). Furthermore, surgical decision making is based on studies which evaluate objective measures of success, with the vast majority of seminal trials not taking into account patient-centered outcomes related to choosing a surgery such as: time off work, return to normal activity, need for caregivers/support persons and patient expectations for what constitutes successful surgery and improvement in quality of life. To fill this knowledge gap, this multi-institutional comparative study between TVH+USLS and MI-SCH+SCP will have three specific aims. First, the study will evaluate 3-year surgical failure rates, assessed at 6-month intervals postoperatively for MI- SCH+SCP compared to TVH+USLS; where surgical failure is defined as presence of at least one of the following: 1) presence of vaginal prolapse defined as a lead point of prolapse beyond the hymen on exam, 2) report of bothersome vaginal bulge symptoms irrespective of prolapse stage, or 3) retreatment of symptomatic prolapse with pessary, or surgery. Second, the study will compare outcomes related to perioperative care and recovery including short-term outcomes: post-operative pain, opioid analgesia use, nausea, fatigue, surgical morbidity and long-term outcomes related to body image, sexual, bowel and bladder function assessed immediate postoperatively at 1 month, and at 6 month intervals thereafter. Finally, the study will involve qualitative interviews of a sub-set of women conducted prior to surgery, and at 3 months and 24 months after surgery. The results of these interviews will be incorporated with objective outcomes to develop a comprehensive, patient-centered approach to the treatment of pelvic organ prolapse.

NIH Research Projects · FY 2024 · 2021-08

Heart failure with preserved ejection fraction (НFpEF) is a syndrome that manifests in approximately 50% of all heart failure patients. The incidence and prevalence of НFpEF is sharply rising in the last years, however most patients remain unaddressed. One of the main reasons is the lack of efficacy of drugs, but the other is that historically HFpEF patients were excluded from many heart failure clinical trials. As a result, current treatment guidelines exist for other heart failure types, but completely lack for HFpEF. This proposal will develop a new class of biomaterial-based immunomodulators which work via targeted modulation of specific immune cells in HFpEF. These cells, namely monocytes and T-lymphocytes, are known to be responsible for HFpEF pathogenesis and their targeting is hypothesized to delay the onset of HFpEF and, possibly, alleviate its common consequence - cardiac fibrosis. We propose to develop such biomaterials and test them in mouse models of HFpEF through consecutive execution of the following Specific Aims: 1) We will synthesize and test these polymeric materials with the goal of developing lead formulations that will effectively tune activation of the immune cells in vitro; 2) We will test lead formulations in in vivo models of immune cell activation and further optimize our lead formulations; 3) We will thoroughly investigate best-performing materials in clinically-relevant mouse models of HFpEF. In addition, we will thoroughly investigate safety, including evaluation of systemic immunosuppression of these materials. Therefore, this proposal is significant, because targeting inflammatory and immune pathways proposed here could provide a promising approach for developing therapeutic options in HFpEF patients who have not yet responded adequately to approved and late-stage investigational treatments.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Hypertension disparities persist and are particularly worst in African Americans (AA). Suboptimal hypertension self-management, including adherence to medication-taking of antihypertensives remains a major public health concern. Poor adherence to antihypertensive medications is estimated to occur in 43-78% of patients, with approximately 50% discontinuation after a year, and worse in AA. Even more alarming, AA older adults may not be prescribe evidence-based practice treatment regimens known to more efficient in the AA population. There is a critical need for behavioral approaches and long-term self-management strategies that are feasible, replicable, and scalable. Mobile Health (mHealth) technologies (mobile phone applications [app], text, video messaging) are promising tools to facilitate behavioral change and sustain self-management. While reports support using mHealth technologies for the management of chronic diseases have grown, there is limited data specific to AA older adults. Other gaps identified in the literature regarding mHealth technology include the lack of being theoretically driven, the clinical/epidemiologic investigations are primarily conducted outside of the U.S. limiting generalizability, and the lack of evidence on long-term sustainability and impact on clinically relevant health outcomes. This R01 application, OPtimizing Technology to Improve Medication Adherence and BP Control (OPTIMA-BP) is testing a technology-based interventions compared to a wait-listed control (WL) in a prospective, randomized controlled trial (RCT) design. OPTIMA-BP includes evidenced-based strategies, web-based education, and behavioral skills training to use a theoretically driven mHealth medication management app in conjunction with a guideline directed treatment regimen and nurse counseling. The aims of this study are: [1] To test the effects of OPTIMA-BP vs. WL on systolic BP and serum high- density lipoprotein cholesterol (HDL) in AA older adults with hypertension in a prospective, RCT format; and [2] To test if the attitudinal/knowledge mechanisms of self-management (hypertension knowledge, self-efficacy, perceived social support) and proximal behavioral target mechanisms (taking medications to reduce systolic BP, diet, exercise) mediate OPTIMA-BP vs. WL’s impact on the primary and secondary outcomes (systolic BP, diastolic BP, health-related quality of life, serum lipids, and at least 62% of the sample with BP <130/80 mmHg) over a 12-month time period. A secondary aim of this study will assess OPTIMA-BP and WL’s impact on the primary and secondary outcomes while controlling for covariates/contextual variables such as age and gender. A supplemental and complementary feature of this proposal is the qualitative evaluation to confirm self- management barriers and perceived strengths or limitations of the intervention, which will inform future refinements should these RCT findings be positive.

NIH Research Projects · FY 2024 · 2021-08

Project Summary Radiation is a mainstay of cancer treatment, yet challenges remain. The long term goal of the proposed research is to transform traditional cancer radiation therapy protocols by including a pre-treatment step involving perturbing the vascular and cellular function of tumors with ultrasound-activated radiosensitizing nanobubbles (NBs). The new paradigm in cancer treatment protocol builds upon a decade of prior work that used commercial microbubbles (MBs) to elicit a radiosensitizing effect. The MB radiosensitization effects are primarily intravascular, with significant endothelial damage incurred. In contrast, in the strategy proposed here, we hypothesize that the NBs will also extravasate into the tumor parenchyma, which will result in significant increases in direct damage to the cancer cells, in addition to the vascular damage. Thus the effect will be both intra- and extra-vascular. The tumors treated in this way will respond better to radiation, lowering the effective radiation dose and decreasing residual surviving tumor. The technique further allows targeting of tumor specific volumes allowing healthy tissues to be spared. We have demonstrated in preliminary studies in vivo that ultrasound-activated NB perturbation of tumors results in a significantly greater enhancement in tumor kill compared to MBs when followed by traditional radiation therapy. This approach could markedly improve existing therapies and reduce the associated side-effects. This is clinically important for prostate cancer treatment where collateral damage and off-target effects are common and lead to years of complications in many patients. Therefore, we propose a set of four specific aims to test, develop, optimize, demonstrate and quantify the efficacy of this novel technique in prostate cancer. Aim 1 will focus on the development of stable, uniformly-sized radiosensitizing NBs. The acoustic and bio-activity of the bubbles will be measured, and baseline biodistribution in tumor bearing mice will be carried out. In Aim 2, the NBs will be tested in combination with radiation in a mouse model of prostate cancer so that treatment parameters can be optimized. In Aim 3, carried out concurrently with Aim 2, we will develop a photoacoustic imaging approach for monitoring early treatment response. This tool will be used to predict therapeutic efficacy and completeness of tumor treatment as soon as 2 hours after the therapy. Finally, in Aim 4, we will test the combination approach in a large (rabbit) orthotopic model of human prostate cancer. We have assembled a multidisciplinary MPI team of investigators with a demonstrated track record of collaborative work in this field. The team includes Dr. Czarnota MD/Ph.D., a physician-scientist and discoverer of the original MB sensitizing approach now in clinical trials, Dr. Michael Kolios Ph.D. is a medical physicist with broad experience in photoacoustic imaging for therapy response and ultrasound physics and Dr. Agata Exner, Ph.D., a biomedical engineer with extensive expertise in formulation and implementation of nanobubbles for imaging and therapy. Members of the team are actively collaborating, have shared publications, grants and projects, and record of technology translation to the clinic. The team will ensure timely completion of the proposed research and rapid translation of the approach to clinical use.

NIH Research Projects · FY 2024 · 2021-08

Abstract. Prostate cancer (PCa) is the most common malignancy and the second leading cause of cancer death in men in the United States. Although surgery and radiation therapy in patients with low risk disease appear appropriate and effective, those with high-risk localized disease almost always become hormone refractory and then rapidly progress. New treatment strategy is urgently needed for patients with high-risk localized prostate cancer, particularly an approach that considers the use of a multimodal approach and that includes both local and systemic therapies. Cytotoxic drugs are broadly used to treat hematological malignancies and solid tumors and, under certain clinical conditions, have changed the natural course of some of these diseases. While effective, due to their intrinsic mode of action, they may also cause significant off-target adverse events that could preclude their full clinical efficacy, possibly resulting in early discontinuation of medication and a consequent increased risk of tumor relapse or recurrence. Alternative approaches to both maintain the effectiveness of chemotherapeutic drugs and minimize systemic toxicity include conjugation of cytotoxic agents to humanized antibodies (also known as Antibody Drug Conjugates, ADCs). The durable clinical responses reported with brentuximab vedotin (SGN-35: Seattle Genetics/Takeda) and trastuzumab emtansine (T-DM1; Roche in partnership with ImmunoGen), which have recently obtained regulatory approval, have profoundly changed the outlook for ADC cancer therapy. These approaches, although showing strong potential, are extremely expensive, and less complex and more cost-efficient methodologies are needed. Here we describe the use of a novel ligand for prostate specific membrane antigen (PSMA, a biomarker for prostate cancer) to target a potent microtubule inhibiting agent, MMAE, and a photodynamic therapy (PDT) agent, IR700, selectively to prostate cancers. The design of this new drug molecule utilizes a prodrug approach and simultaneously delivers two drugs selectively to prostate cancer. By selective delivery of two drugs with different therapeutic mechanisms to cancer cells, improved antitumor activity with less toxicity is expected. The reduction in toxicity is expected due to anticipated drug synergy (requiring lower drug doses), site specific prodrug activation, and rapid clearance of the drug molecule, preventing off target delivery. This molecule will be developed using two animal models of prostate cancer, heterotopic human prostate cancer in mice and spontaneous prostate cancer in companion dogs. Both MMAE and IR700 therapy have been noted to stimulate immune response against cancer and we will preliminarily tested this in the immunocompetent companion dogs. Dog pathology and physiology of prostate cancer is very similar to humans and dogs are often used in drug development trials. Since efficacy trials in mice are not predictive of human results, efficacy studies in dogs will substantially encourage clinical translation of the developed agent.

NIH Research Projects · FY 2025 · 2021-08

The overarching goal of this proposal is to develop precision therapy for PIK3CA-mutant colorectal cancer (CRC). Phosphatidylinositol 3-kinases (PI3K) are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit. The PI of this application co-discovered that PIK3CA, which encodes p110α, is frequently mutated in a variety of human cancers, including ~30% of CRC. Most PIK3CA/p110α oncogenic mutations occur at two hot spot regions, one in the helical domain and the other in the kinase domain. Nearly half of all p110α mutations are located in the helical domain. Increasing evidence suggests that the helical domain and kinase mutations exert their oncogenic function through distinct mechanisms. For the helical domain mutations, we discovered that the oncogenic signal is transduced by two unique pathways. We previously found that the p110α helical domain mutant protein directly associates with IRS1 independent of p85 to activate PI3Kα-AKT. Now, our preliminary studies demonstrate that p85β disassociates from the p110α helical domain mutant protein complexes and translocates into the nucleus. The nuclear p85β stabilizes EZH1 and EZH2, two enzymes that catalyze histone H3K27 trimethylation. Remarkably, we found that a combination of EZH inhibitor GSK126 with a p110α-specific inhibitor Alpelisib induced tumor regression of CRCs harboring a PIK3CA helical domain mutation. Thus, we hypothesize that nuclear p85β promotes oncogenic functions of p110α helical domain mutations and that simultaneous inhibition of both nuclear p85β function and p110α kinase will be an effective approach to treat tumors with a helical domain mutation. Two aims are proposed to test the central hypothesis: 1) elucidate the mechanisms by which nuclear p85β promotes oncogenic functions of PIK3CA helical domain mutations; and 2) determine the efficacy of the combination of GSK126 and Alpelisib using a panel of CRC patient-derived xenografts with a PIK3CA helical domain mutation. Successful completion of our studies will demonstrate the combination of GSK126 and Alpelisib as an effective treatment for CRCs with PIK3CA helical domain mutations in preclinical models and lay a solid foundation for future clinical trials. Moreover, our studies uncover p85β nuclear translocation as a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic functions.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract The idea that information processing depends on neuronal firing rate (rate coding) has long been a central dogma in neurobiology. However, other non-canonical coding schemes (temporal and “analog” codes) have been proposed to carry meaningful information and be more computationally powerful than rate coding. Importantly, the field has lacked powerful genetic model systems to disentangle non-canonical coding processes, and I addressed this gap by defining two neural circuits in Drosophila that can be used to study temporal and analog codes. I found that temporal coding underlies the circadian regulation of sleep in the Drosophila DN1p clock neurons, whereas analog and potentially “hybrid” (analog + spiking) codes are used to achieve axon-specific hunger processing in Drosophila DA-WED feeding neurons. As a model system to understand how spiking temporal codes impact molecular signaling and behavior, we will focus on Drosophila DN1p clock neurons having specific spiking patterns to control sleep quality through a novel form of synaptic plasticity, SPDP (Spike Pattern Dependent Plasticity). To examine the molecular process of SPDP formation, we will first characterize essential elements of the temporal structures within irregular spiking patterns in DN1ps, as well as identify their biophysical origins. Next, we will investigate molecular mechanisms that act downstream of presynaptic spiking patterns to transform electrical signals into biochemical responses. We will also leverage the power of Drosophila genetics to delineate the entire molecular pathway required for SPDP in DN1ps synapses. As a model system to understand how nonspiking neuronal codes impact signaling and behavior, we will focus on Drosophila DA-WED feeding neurons having local plasticity to control protein hunger behavior. Neural coding paradigms have generally focused on “digital” all-or-none spike-based models. In mammals, pure “analog” coding occurs in the retina, but recent work has shown that analog signaling modulates spike-based signaling (“hybrid” coding) in the hippocampus and cortex. However, the function of these codes in neural plasticity and behavior remains unclear. We recently discovered that the “protein coding” axonal branch, but not the “sugar coding” axonal branch, exhibits sub-threshold membrane potential fluctuations of DA-WED feeding neurons, following mild protein deprivation. Following severe protein deprivation, such analog signaling interacts with spiking events to generate “hybrid” processing to achieve stronger and longer-lasting protein feeding behavior. Thus, we will study the molecular processes mediating “analog” and “hybrid” signaling and how “hybrid” codes may underlie localized branch-specific plasticity. In conclusion, these studies using Drosophila DN1p clock neurons and DA-WED feeding neurons should elucidate fundamental principles for non-canonical neural codes, determine the role of these neural codes in long-lasting behaviors and plasticity, and identify their underlying molecular mechanisms.

NIH Research Projects · FY 2025 · 2021-08

Project Summary We seek to understand how the physiochemical environment of the extracellular matrix affects the diffusion, structure, and folding of proteins. The extracellular matrix is spatially heterogeneous at nanoscales and can change over time. But current imaging methods are limited in spatial and temporal resolutions, while biochemical techniques are designed for model, pristine solutions that differ from the complexity of the extracellular matrix. Therefore, how proteins function locally in the extracellular matrix is an underexplored area in biophysics. We propose to understand how proteins function and interact in the complex extracellular matrix environment, and how the nanoscale variation in the chemical and physical composition of this environment can locally change protein dynamics. To achieve this, we will use a correlation-based super-resolution microscopy developed by our lab that can access the time- and spatial-scales of protein dynamics within the extracellular matrix. The goals of this proposal are to: 1) identify how the nanoscale chemical and steric properties of the extracellular matrix control protein diffusion, structure, and folding; 2) determine how cells tune the dynamics of proteins by changing the extracellular matrix; 3) develop instrumental and analysis methods that access the spatiotemporal regimes required to understand protein function in the extracellular environment. Achieving our goals will reveal the extracellular matrix and protein dynamics that drive biological regulation and will provide researchers with methods to understand and potentially control extracellular delivery of signaling proteins or therapeutics. Overall, an entirely new area of biology – the extracellular matrix - will be explored by super-resolution microscopy; the findings can be broadly applied to other biomolecules, cells, and tissues of relevance to fundamental biophysics, drug-delivery/therapeutics, and disease.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Advances in high throughput sequencing have already revealed millions of protein coding variants within human exomes, and there are many millions of additional differences that likely exist but have not yet been observed. Many of these variants likely play important roles influencing human health, but we lack the clinical data required to associate each variant genotype with their corresponding phenotypes. This disconnect is oftentimes referred to as the variant interpretation problem. Genetic experiments in model systems play a critical role in uncovering the effects of protein coding variants, but traditional approaches typically test variants one-by-one and will never address this glut of uncharacterized variants. Multiplex genetic assays capable of simultaneously testing complex variant libraries have the required throughput, but these approaches are still in their developmental infancy, and improvements are needed to increase the capabilities, costs, efficiency, and usability of these techniques to successfully address this problem. Harnessing a palette of synthetic biology tools centered around the highly efficient Bxb1 bacteriophage DNA recombinase, I developed a user-friendly, highly customizable platform for expression of complex variant libraries within individual cultured human cells. I previously paired this expression system with a highly generalizable assay that identifies variants that are loss-of-function due to an reduced intracellular steady-state abundance. I applied this assay to comprehensively study variants in four disease- related proteins, and more collaborative projects are still in progress. Unfortunately, these methods alone will not address the problem, and more orthogonal approaches are needed to tackle the millions of uncharacterized disease-relevant variants that exist within people. The goal of this proposal is to build the next set of fundamental biotechnologies needed to enable more high-throughput characterizations of protein variants. The individual directions described are each highly generalizable and can be reapplied to study large swaths of the proteome with only slight modification. Immediate directions include a functional complementation system to study essential genes, a fluorescent transcriptional reporter system to study perturbations to intracellular signaling pathways, and a barcoded ORFeome collection to identify genes that modulate phenotypes of interest when they are overexpressed. A major purpose of these technologies is to facilitate adoption by other research groups, especially those that are experts in other biological fields. These developments, along with the data we generate in the process of demonstrating their utility, will directly address the variant interpretation problem while also uncovering previously hidden biology underlying cancer-related molecular mechanisms critical to cell function.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The CHCHD10 gene coding for a mitochondrial protein is mutated in familial and sporadic Frontotemporal Dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), and mixed FTD-ALS. The estimated prevalence of CHCHD10 mutations is 7.7% among FTD in the Chinese population and 0.68-2.6% among FTD-ALS patients of European descent, making CHCHD10 the second most frequently mutated gene in FTD and FTD-ALS. We know that the FTD-ALS CHCHD10S59L mutation and the ALS CHCHD10R15L mutation promotes CHCHD10 aggregation and mitochondrial dysfunction. However, as only 1 patient with CHCHD10 mutation (ALS-linked CHCHD10R15L) and no FTD patient with CHCHD10 mutation has come to autopsy, we do not know the pathological signatures of CHCHD10-driven pathogenesis and to what extent such signatures are present in sporadic diseases (i.e. FTD, FTD-ALS, AD). As misfolded proteins tend to clog and inhibit the proteasome, such misfolded and aggregation-prone proteins are frequently translocated into mitochondria as an alternative pathway for proteostasis. We recently generated transgenic (Tg) mice expressing wild type (WT) CHCHD10WT, CHCHD10R15L, or CHCHD10S59L driven by the neuronal mouse PrP promoter, which show clear pathophysiological phenotypes. By taking advantage of mouse models and human postmortem tissues together with molecular, biochemical, histochemical, proteomics, electrophysiological, and behavioral toolsets, this proposal will test the overarching hypothesis that the loss of endogenous CHCHD10 (as seen in sporadic ADRDs) and FTLD/ALS-linked CHCHD10 mutations drive diverse pathological signatures resulting from disruptions in mitochondrial proteostasis and autophagic clearance of proteotoxically challenged mitochondria, and that restoration of WT CHCHD10 represents a viable strategy to mitigate proteotoxic burden and disease outcomes. Aim 1 will define the role of wild type CHCHD10 in mitigating pathological phenotypes in vivo. Aim 2 will identify and validate the neuropathological signatures of FTLD/ALS-linked CHCHD10 mutations. Aim 3 will determine the role of mutant CHCHD10 in mitophagy flux in vivo.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Particulate matter air pollution <2.5µm (PM2.5) is the leading environmental risk factor globally, contributing more to global mortality than AIDS, malaria, tuberculosis and wars/famines combined. The health risks associated with PM2.5 are predominantly from cardiovascular (CV) causes, with data supporting a major public health impact due to disorders such as type 2 diabetes. Building on the substantial successes of our research program, that in large part have provided the foundational basis for our understanding of mechanisms of PM2.5-induced cardiometabolic disorders, we propose an innovative framework of translational investigations. Our new data provide compelling links between PM2.5 exposure and circadian rhythm (CR) disruption through epigenetic reprogramming, resulting in metabolic dysfunction and insulin resistance. In accordance with the NIEHS Translational Research Framework, we propose an ambitious plan encompassing 3 goals over 8 years that will encompass harmonized studies involving concentrated ambient PM2.5 exposures (animal) and human intervention trials across the ambient exposure spectrum, predicated and supported by results from genome wide association and mendelian randomization approaches, that will identify pathways of air pollution mediated CV risk. Leveraging on-going translational clinical trials of air pollution reduction using personalized strategies in vulnerable populations in Beijing, and through one new study involving mitigation of air pollution (ITS-MY-AIR), we will evaluate the impact of exposure reduction on CR disruption/sleep and metabolic endpoints. Collectively, results from this project will help shed important new light on air pollution, its impact on CR and cardiometabolic disorders and provide influential data on protective effects of personal intervention strategies, that together could change public health policy.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Degeneration of brain or spinal cord neurons is associated with many untreatable neurological disorders and leads to a gradual decline in cognitive and motor function, and eventual death. While more prevalent in aged individuals, neurodegenerative disease onset is variable and can begin in childhood. There are no effective therapies for the majority of patients, likely due to our limited understanding of disease etiology. Inherited neurodegenerative disorders are commonly caused by genetic mutations in RNA binding proteins that regulate RNA biogenesis. In order to develop therapeutics for this class of disorders, it will be critical to understand how RNA binding proteins function in normal and diseased states, and whether molecular changes are amenable to correction. Our preliminary data suggests mRNA processing may be significantly affected in cases of inherited childhood motor neuron degeneration. To test our hypothesis that mRNA processing defects cause pediatric motor neuron disease, we have created human stem cell- and animal-based models to correlate molecular changes with disease pathology. We will apply high-throughput sequencing, electrophysiology, histology and behavioral approaches to our novel disease models to pinpoint pathogenic transcriptional mRNA isoforms expressed as a consequence of the RNA binding protein mutation as well as test a candidate targeted gene- based therapy.

NIH Research Projects · FY 2025 · 2021-06

Abstract Natural killer (NK) cells are lymphocytes that are capable of killing malignant cells without antigen-specific receptor recognition. Due to this property, NK cells have been utilized for adoptive immune cell therapy for a variety of malignancies. Despite limited success for hematologic malignancies, the therapeutic potential of NK cell therapy for cancer has been limited by the insufficient cytotoxic activity of NK cells particularly for solid tumors. Due to the ability of NK cells to specifically target cancer cells and avoid killing of normal cells, we hypothesize that enhancing the cytotoxic activity of NK cells can lead to enhanced efficacy of NK cell therapies for cancer. As the molecular mediators regulating NK cell cytotoxic activity are still not fully characterized, we will conduct a small molecule high throughput compound library screen to identify novel mediators of NK cell cytotoxic activity. The purpose of this work is to identify small molecules that can be further developed for therapeutic purposes as well as to reveal important probes to elucidate mechanistic insights into NK cell activity that may enable future targeted approaches. In the first aim, we will identify compounds that enhance NK cell killing of ovarian cancer cells through a HTS of a diverse 300k small molecule collection. In the second aim, the primary hits will be tested in a series of secondary assays to identify the most promising hits. The most promising hits will then undergo biological characterization in vitro and in vivo. It is hoped that this work will lead to the identification of promising approaches to improve NK cell therapy for cancer patients.

NIH Research Projects · FY 2025 · 2021-05

Abstract Biomaterials-based strategies to modulate the immune responses has generated tremendous interest in the past decade. Notably, biomaterials can not only be used for delivering drugs (synthetic or biologics) but by themselves can modulate the function of different cells. Recently, we have demonstrated that the metabolite alpha-ketoglutarate (aKG) can be polymerized, and these polymers by themselves are able to suppress activation of dendritic cells (DCs – forms the bridge between innate and adaptive immune system). Interestingly, our preliminary data also demonstrates that delivery of PFK15, an inhibitor of PFKFB3 enzyme (a key step in glycolysis) downregulates CD86 (co-stimulatory molecule) but maintains MHC-II (stimulatory antigen presenting molecules) on DCs. Notably, glycolysis can control the function of activated DCs. Therefore, glycolysis-inhibition mediated prevention of DC activation and simultaneous antigen expression, can lead to antigen-specific immunosuppression responses. However, systemic inhibition of glycolysis has inherent toxicity (clinical trials) associated with it, and have regulatory hurdles for clinical use. Therefore, the main goal of this R01 program is to develop drug delivery vehicles that can deliver glycolysis inhibitors and antigens locally to DCs, which will then systemically suppress inflammation. The central hypothesis of this proposal is that co-delivery of antigen and glycolytic inhibitor will induce DC tolerance and generate peripheral antigen-specific suppressive T-cells, which will then promote reversal of tissue inflammation. This strategy will be tested in a rheumatoid arthritis animal model. This hypothesis will be tested by performing experiments in the following aims: AIM 1: Test if paKG formulations can generate long-term remission of RA by maintaining metabolic homeostasis in joint tissues. AIM 2: Determine the effect of paKG formulations on cells associated with arthritic tissue. AIM 3: Test the ability of paKG formulations to prevent progression of RA in K/BxN mice AIM 4: Develop scaled paKG formulations for safety/toxicity profiles. This research will be an important foundation in the development of technologies based on metabolic modulation of immune cells for autoimmune disorder treatment. The results from this project will generate a sustained release platform, which after application can prevent the progression of RA, or even reverse the damage.

NIH Research Projects · FY 2024 · 2021-05

PROJECT SUMMARY/ABSTRACT: A number of candidate therapies such as CRISPR-Cas9 and gene silencing require the efficient delivery of functional nucleic acids to the cell cytosol and nucleus. Unfortunately, such therapies currently lack proper delivery mechanisms, precluding their widespread applicability. Self- assembled deoxyribonucleic acid (DNA) nanoparticles have shown potential as minimally cytotoxic therapeutic carriers in cancerous and other in vitro and in vivo models. While evidence suggests that DNA nanoparticles-based drug carriers can be taken up by mammalian cells via endocytosis, it is unknown how these DNA nanoparticles can overcome the fate of endocytosis-triggered degradation to reach the cytosol and, once there, can controllably maintain stability. With the enabling science explaining their behavior and mechanisms of controlling their stability in the cell cytosol it will be possible to make bold advances in engineering therapeutic delivery systems. To that end, the proposed work has two overarching scientific payoffs. Payoff 1, induce endosomal escape and enhanced cytosolic accessibility of DNA nanoparticles by the integration of calcium in their assembly process. Payoff 2, identify the rate of breakdown and mechanisms of stabilization of DNA nanoparticles in different types of cell cytosols. Innovative technologies will be the foci of the PI's training program and will be implemented to achieve the project goals, namely, multi-step Förster resonance energy transfer spectroscopy for high-resolution tracking of DNA nanoparticle inside the cell and in vitro cell microinjections enabling study of these nanoparticles directly in the cytosolic environment. First, a DNA origami based nanotube will be tested for structural stability in calcium-supplemented buffer. Thereafter, the nanotube will be used as a carrier for the delivery of functional RNA molecules to representative fluorescent protein-expressing cells and checked for its cytosolic reachability and efficacy in protein regulation after undergoing endocytosis. Second, small (20 nm) DNA nanoparticles with branched architecture and non-canonical nucleic acids will be embedded with multi-step FRET reporters for measuring structural integrity. These DNA nanoparticles will be microinjected into live cells cytosolic region and their breakage be determined. Last, the cytosolic stability of these DNA nanoparticles will be correlated with different types of mammalian cells with known cytosolic variability (tumor, immune, and other cell types) in order to map the role of structurally diverse DNA nanoparticles in targeting cells with different physiologies. The PI will also receive training in rigorous analysis of in vitro research, lab management, and the prolific grant writing process, which will facilitate their transition to an independent research program. Outcomes of this project will pave the way towards developing more bio-compatible delivery systems, specifically for functional nucleic acid therapeutic agents that are vital in the cell cytosol.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY This proposal addresses a significant public health question: Does diabetes, the 3rd leading cause of death in the United States (US), impact suitability of donor corneal tissue for transplantation? This question takes on increasing urgency as recent eye bank data suggests donors with diabetes now comprise about 30-35% of the cornea donor pool, a 50-72% increase in just over a decade. The impact of diabetes on keratoplasty outcomes remains unknown, with conflicting evidence from secondary or retrospective analyses of multiple clinical studies. Previous large clinical studies did not show a diabetic donor effect on penetrating keratoplasty (PKP) and Descemet membrane endothelial keratoplasty (DMEK) graft success, yet our recent Cornea Preservation Time Study (CPTS) found the diabetic donor adversely affected graft outcomes following Descemet stripping automated endothelial keratoplasty (DSAEK). Although current standard of care is to use diabetic donor corneas for all types of keratoplasties, some eye banks and surgeons are increasingly avoiding them for DMEK. As both the diabetic donor population and DMEK demand increases, a definitive superiority study evaluating effect of donor diabetes status on graft outcomes will allay and/or define these concerns. The Diabetes Endothelial Keratoplasty Study (DEKS) will address these important questions through a prospective masked clinical trial enrolling 1420 participant-eyes at 30 clinical sites and 15 eye banks across the US. The DEKS will determine if the 3-year graft success rate following DMEK performed with corneas from donors without diabetes is superior to the graft success rate with corneas from donors with diabetes. It will also determine if the 3-year central endothelial cell loss (ECL) after DMEK with corneas from donors without diabetes is less than the central ECL when corneas from donors with diabetes are used. Lastly, the DEKS will explore the relationship of donor diabetes severity, as measured by eye bank-determined diabetes risk categorization scores, post- mortem HbA1c, and skin advanced glycation endproducts and oxidation markers, with DMEK graft outcomes 3 years postoperatively in corneas from diabetic donors. The DEKS could have a major impact on the targeted use of corneas from an increasing number of donors with diabetes with a range of disease severity in a donor pool that must continue to expand to meet the clinical demands of an aging population and DMEK growth.

NIH Research Projects · FY 2026 · 2021-05

Fatigue is a frequently reported complaint of adolescents and is linked to a wide range of adverse health, behavioral, and functional outcomes. Variation in fatigue prevalence exists, especially among families that experience challenges in socio-environmental conditions. Poorer sleep quantity and quality also occur among families facing similar challenges in socio-environmental conditions. Because of fatigue’s strong link with inadequate sleep, our understanding of fatigue will benefit from greater awareness of its relationship with variation in sleep, especially among early adolescents, when notable differences in sleep duration and timing emerge. Numerous social-environmental factors in individual, household, and neighborhood levels may serve as risk and resilience factors shaping the experience if sleep and fatigue. Yet, the relative contributions of these factors are yet unclear. Moreover, the association between inadequate sleep and fatigue is obscured by another common teen complaint: daytime sleepiness. The relationship between fatigue and sleepiness, and their differential effects on adolescent health and functioning are yet unclear. Our study purpose is to identify key mechanisms through which socio-environmental factors influence variation in sleep domains, and sleep domains’ associations with fatigue, daytime sleepiness, and functioning in early adolescents. We posit that differences in socio-environmental factors such as household organization and socioeconomic status drive variation in sleep domains across different adolescent populations. Guided by a community advisory board, and using a novel smart phone/sensor technology developed in our pilot research on adolescent sleep, we propose a home-based study among adolescents in Cleveland, OH to (a) Identify socio-environmental factors responsible for variation in adolescent sleep; (b) Determine the effects of observed sleep variation on fatigue, daytime sleepiness, and functioning; and (c) Disentangle the effects of sleepiness from fatigue. Using a mixed-methods design and sample of 350 adolescents and caregivers balanced by a range of study variables, linear mixed modeling will assess associations between baseline (time-invariant) and nightly (time-varying) measures of key mechanisms with that of sleep quality and quantity, fatigue, sleepiness, and functioning over a 2-week period. A qualitative study component will focus on how household/neighborhood factors shape sleep, the teen experience of sleepiness vs. fatigue, and self-management strategies to address problematic sleep and fatigue. The study is innovative: it simultaneously investigates multiple potential mechanisms driving variation in teen sleep and uses novel technology to better measure the context of teen sleep. Expected results will be significant: identifying drivers of sleep variation is vital to improve sleep, reduce fatigue and sleepiness, and improve adolescent health, functioning, and quality of life. Moreover, disentangling fatigue from daytime sleepiness is needed because their clinical implications and treatment differ.

NIH Research Projects · FY 2025 · 2021-05

Project Summary / Abstract Recent research indicates that when ~808 to 830 nm light is applied in immediate juxtaposition to target neurons or axons (within mm) through invasive techniques, C and Aδ fibers that convey pain-related information can temporarily and reversibly be “turned off” without affecting the functionality of the larger A fibers. If further developed, this photobiomodulation (PBM) effect has exciting possibilities as an implantable device-based treatment for various chronic pain syndromes, including neuropathic pains. This project takes important steps to develop fundamental and mechanistic understanding, and to provide a foundation for translation. In terms of fundamental and mechanistic understanding – first, the effect of PBM dose and wavelength on axonal block (in an ex vivo peripheral nerve preparation) and nociceptive response (in an in vivo rodent pain model) will be rigorously characterized. These data will provide important mechanistic insight and translational value. Second, the role of observed microtubule destabilization and the resulting axonal varicosities will be explored as contributors to the mechanism of the independently-observed action potential block. We will determine whether or not there is a correlation between effect size (functional data) and degree of microtubule instability (confocal microscopy and electron microscopy data). Computational models will be used to evaluate the effect of axonal varicosities on action potential propagation. Finally, the effect of pharmacological microtubule (de)stabilizers on PAB dose will be assessed. In terms of translational activities – the project includes development of pre-clinical-grade systems that allow PBM at the nerve to be applied chronically with the ultimate goal of demonstrating that chronic PBM can provide a persistent and profound analgesic effect in a large animal pain model (porcine). A fully implantable system based on an existing commercial neurostimulator will enable PBM to be delivered over extended periods of time. A percutaneous system will require repeated interventions over time (e.g., weekly interventions on the order of minutes), but will enable use of higher peak powers not achievable with the fully implantable system. The systems will be used in a porcine pain model (peripheral neuritis) that better mimicked the human response to pharmacological interventions than rodent models have been able to do. The pre-clinical studies will include a 30-day pilot study followed by a 6-month study in minipigs. In summary, this project will expand fundamental understanding of PBM-induced axonal block with an eye toward translational devices suitable for the treatment of chronic pain.

NIH Research Projects · FY 2024 · 2021-04

− transporters (NCBTs, members of the SLC4 family) play critical roles in transepithelial HCO−3 Na+-coupled HCO3 transport, whole-body pH regulation, and intracellular pH (pHi) regulation. In this Multi-PI R01, the team will exploit powerful techniques, many developed in their respective laboratories, to elucidate molecular transport mechanisms of NCBTs, which are especially important in the kidney. These tools include surface pH (pHS) measurements, out-of-equilibrium (OOE) CO2/HCO3− solutions, macroscopic mathematical modeling (MMM) of acid-base transport in a single cell, and state-of-the-art molecular dynamics (MD) simulations of interactions between the substrates and the transport molecule, or of CO2 conduction through the NCBTs. In a multidisciplinary approach, the team will answer 2 major questions. Aim 1: Do all NCBTs carry some form of – ion pair—whereas other SLC4 “HCO3− ” transporters actually carry =—arriving or departing as the NaCO3 CO3 HCO3− per se? By monitoring pHS in voltage-clamped oocytes the team will test whether SLC4 family members − . They will address the same question in perfused proximal tubules (PTs) from wild- = ” vs. HCO3 transport “CO3 – binding to the KKMIK region type (WT) and NBCe1-A/D knockout mice. They also test the hypothesis that NaCO3 of TM5 is a rate-limiting step for NBCe1-A transport. Using MD, the team will identify/model outward-facing, occluded, and inward-facing conformational states of NBCe1, NBCn1 and AE1, and identify potential interaction sites –, =, −, Cl−, for NaCO3 Na+, CO3 HCO3 and and use MMM (3D reaction-diffusion simulations) to assess physiological data. Finally, in an iterative process, the team will assess single nucleotide polymorphisms (m- = ” vs. HCO−3 SNPs) as well as other mutations suggested by MD studies, prioritize them, and evaluate for “CO3 transport using physiological assay, interpret using MD and MMM, and suggest new mutations. Aim 2: Do all = ” transport, NCBTs conduct CO2 whereas other SLC4 transporters do not? Having presumably committed to “CO3 evolution faced the challenge of translocating the second carbon atom, ultimately derived from 2×HCO3−. The team will use electrophysiological techniques and a novel neutral buoyancy assay (NBA) to ask whether all NCBTs conduct CO2, whereas other SLC4 members do not. In perfused PTs from WT and NBCe1-A/D knockout mice, they will ask if NBCe1-A conducts CO2 in PTs. The team will use MD to identify potential CO2 pathways through NBCe1, NBCn1, and AE1 as a negative control. MMM will assess the physiological data. Finally, in an iterative process, the team will process m-SNPs and other mutations as outlined in Aim 1, but now for effects on CO2 conduction. The research will reorganize our thinking of NCBT function, providing valuable insight into the pathogenesis of proximal renal tubular acidosis (pRTA) and other maladies associated with NBCe1 (e.g., migraine, ocular and dental abnormalities, suicidal ideation), other NCBTs (e.g., hypertension, breast cancer, epilepsy, autism). The systematic analysis of m-SNPs may provide insights into previously unrecognized “NCBT- opathies.” The work also will have broader impact by elucidating physiological acid-base surface chemistry and = ”. − vs “CO3 for the first time permitting on to distinguish unambiguously among the transport of H+ vs HCO3

NIH Research Projects · FY 2025 · 2021-04

Increasing evidence indicates that inflammatory bowel disease (IBD) is considered an intestinal barrier disorder, whereby epithelial dysfunction is central to disease pathogenesis. Single nucleotide polymorphisms (SNPs) in gasdermin B (GSDMB), a gene known to be predominantly expressed in the epithelium of the GI tract, are associated with increased susceptibility to IBD. GSDMB belongs to a family of structurally-related pore-forming proteins, i.e., gasdermins (GSDMs), primarily known for their central role in programmed cell death, or pyroptosis. Indeed, GSDMB can be cleaved by Granzyme A (GzmA), whereby the released N-terminal domain (GSDMB- NTD) oligomerizes to form pores in the plasma membrane, thus mediating pyroptosis. GSDMB-NTD also exhibits direct microbicidal activity, targeting gram-negative bacteria, while sparing host epithelial cells. Our group showed that in its full-length form, GSDMB exerts crucial, non-lytic functions within intestinal epithelial cells (IECs) to control mucosal wound healing, and importantly, GSDMB is not only increased in IBD patients relative to healthy controls, but IBD-associated mutant GSDMB (GSDMBMut) confers dysregulated IEC function(s). Numerous GSDMB protein isoforms have been described, and were thought to account for the aforementioned functional differences. Several papers published in 2023 reported the structural and biochemical analyses of GSDMB, and came to the consensus that a singular exon within its interdomain linker region (i.e., Exon 6) dictates the pore-forming, pyroptotic activity of GSDMB upon GzmA-mediated cleavage and activation. Despite these advances, there remains a significant knowledge gap in resolving the relevance and significance of GSDMB isoforms during health vs. disease states. Our preliminary findings indicate a GI-specific pattern of GSDMB transcript expression in gut mucosal biopsies with a marked shift in IBD patients. Genomic analysis further demonstrates that the rs11078926 GSDMB SNP not only alters splicing and isoform expression, but may potentially contribute to IBD pathophysiology. Furthermore, much attention has recently focused on GSDM regulation by post-translational modifications, specifically S-palmitoylation, leading to differential cellular function(s). Our preliminary data also suggest that cysteine modifications, but mainly by S-nitrosylation, plays a critical role in regulating GSDMB-driven IEC activities. Taken together, the central hypothesis of this proposal is that GSDMB isoforms are differentially expressed in IECs of control vs. IBD patients, whereby normal vs. defective epithelial function is determined in an isoform-dependent fashion and impacted by carriage of GSDMBMut variants; furthermore, IEC-derived GSDMB is differentially regulated by S-nitrosylation that subsequently impacts GSDMB-dependent IEC activities. The overarching goal of the present proposal is to identify and characterize clinically-relevant GSDMB isoform(s) and determine how their regulation can differentially affect epithelial function. This information will not only provide insights into the role of GSDMB in IBD pathophysiology, but aid in the design of novel therapeutics targeting GSDMB-dependent IEC function.