University Of Missouri-Columbia

NIH Research Projects · FY 2025 · 2022-08

The proportion of the US population of ≥65 years of age is expected to reach 22% by 2050. Healthcare innovations have played a crucial role in improving the health and well-being of the elderly population. Nevertheless, a major gap persists between academia and industry that must be bridged to drive innovation and commercialization of new drugs, devices and technologies to combat age-related disorders, including Alzheimer’s disease (AD) and Alzheimer’s Disease related dementia (ADRD). Graduate students and post-doctoral trainees pursuing advanced training in age-related diseases constitute a vital work force capable of filling this gap; however, current training programs do not provide the business and communication skills to needed to empower these trainees to fill this void. The overarching objective of the proposal is to grow the scientific work force promoting healthy aging through the creation of Biomedical Entrepreneurship Training for Aging (BETA) program. MU is ideally suited to serve as the hub of an educational program focusing on graduate students and postdoctoral fellows pursuing careers in aging-related disciplines, as it co-localizes on one campus a school of medicine, college of engineering, school of health professions, college of nursing, school of journalism, and the comprehensive UM Health System. MU has outstanding programs that support entrepreneurial education, including the Life Science Innovation &Entrepreneurship Graduate Program, the Midwest Biomedical Accelerator Consortium, an NIH-funded Research Evaluation and Commercialization Hub (REACH), a Coulter Biomedical Accelerator, and the MU Life Science Business Incubator. The UM System, with >70,000 students and hundreds of graduate programs, provides a robust source of BETA trainees, including the MU Interdisciplinary Neuroscience Graduate Training Program, University of Missouri-Kansas City (UMKC) Doctoral Program in Applied Cognitive &Brain Sciences, University of Missouri-St. Louis (UMSL) Behavioral Neuroscience PhD program, and the Missouri S&T (MS&T) Graduate Program in Biological Sciences. BETA leverages the commercialization and educational expertise of the statewide UM System to create a network that recruits and mentors and provides them with the biomedical innovation and communication skills needed to become highly productive members of the scientific work force devoted to improving the health and wellbeing of the aging population.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT While hormonal contraceptive methods are highly effective, they are not the method of choice for many women. The National Health Statistic Report indicates that on-demand over-the-counter (OTC) contraceptives (i.e., spermicides and condoms) are the products of choice for many women, especially teenagers. Unfortunately, OTC methods have high failure rates. As a result, teenage pregnancies contribute to more than 70% of unintended pregnancy rates in the United States. Therefore, there is a critical need for new and improved on- demand methods for women. In this proposal, we will explore a new female contraceptive method that will target two processes: 1) blocking post-ejaculated semen and 2) inhibiting sperm motility in the female reproductive tract. After ejaculation, semen changes from a gel-like to a watery consistency via the prostate-derived serine protease called kallikrein 3 (KLK3). Therefore, the central hypothesis for this application is that pharmacologic blockade of semen liquefaction provides a contraceptive effect by blocking sperm from reaching the site of fertilization. Our previous work showed that a serine protease inhibitor effectively blocked those two processes and prevented pregnancy in female mice. Based on these findings, it is hypothesized that small molecule KLK3 inhibitors with targeted activity can be developed for use as novel, non-hormonal, fully reversible contraceptives for women. In this application, a team of investigators will test the action of small molecule KLK3 inhibitor(s) in human semen and sperm function, pregnancy prevention in female rhesus macaques, and will identify novel small molecules inhibitors of KLK3 in addition to the prototype inhibitor. The hypothesis will be tested in three Specific Aims: 1) to characterize the contraceptive potential of specific KLK3 inhibitors in human semen, 2) To evaluate the cellular toxicity of specific KLK3 inhibitor and “hit” compound(s) in human cervical/vaginal cells and 3D vaginal cultures, 3) to characterize the contraceptive efficacy and pharmacodynamics/kinetics of specific KLK3 inhibitor in female rhesus macaques, and 4) To identify novel drug- like KLK3-specific inhibitors from DNA-encoded chemical libraries. Upon completion of this study, we will be able to demonstrate whether serine protease inhibitor can be developed as the first contraceptive product (i.e., film/sponge) that prevents semen liquefaction as well as inhibits the sperm function.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Shigella causes bacillary dysentery with high worldwide morbidity and childhood mortality with complications that include cognitive and developmental impairment in children suffering multiple diarrheal episodes each year. Shigella’s main virulence factor is its type III secretion system (T3SS), which is used to deliver effector proteins into host cells to promote pathogen entry. T3SSs are shared by many Gram negative pathogens with the injectisome comprising a(n): 1) external needle and tip complex for delivering translocators and effectors; 2) basal body that spans the bacterial envelope; and 3) cytoplasmic sorting platform (SP) that energizes and controls secretion. We pioneered visualizing the SP by cryo-electron tomography (cryo-ET) and since then we and others have identified the components of the SP. We now propose studies to explore structural differences between the “on” and “off” secretion states for the in situ injectisome with parallel biochemical analysis of the SP sub-assemblies and the first visualization of the Shigella injectisome-host membrane interface in situ. We will use cryo-ET methods to view the SP pods at a 1-nm or better resolution and then use biochemical and molecular methods as we develop models of assembly and function. In our investigation of the SP, we will explore the movement of protein domains at the SP interface with the inner membrane ring. In parallel, we will extend our study to examine another important cytoplasmic component of the injectisome - the export gate nonamer formed by MxiA, which undergoes structural rearrangements in the absence of the SP. We will also exploit a system we’ve generated for trapping different secretion substrates within the in situ injectisome so that we can determine how the overall structure compares for three different states. These are the “on” injectisome (ipaD null strain) and two forms of “off” injectisomes (mxiH null strain and effector-blocked strains), which will provide functional insight into SP and export gate communication and association. We propose three complementary aims: 1) Define the makeup, intermediate states and structural requirements at the SP/inner-membrane ring (IR) interface that allow SP assembly and guide type III secretion. 2) Correlate export gate structural features with secretion status using complementary cryo-ET and molecular methods; and 3) Identify the structural changes associated with trapping substrates within the in situ injectisome and begin generating the first high-resolution picture of the injectisome-host membrane interface in situ using cryo-ET. Improved cryo-ET methods provide an unprecedented view of substructures within the Shigella injectisome in situ to reveal elements that cannot be studied using purified needle complexes and this is reflected in the preliminary data presented here. We can now visualize these sub-structures, target them for molecular analysis and purify them for in vitro biochemical and biophysical analysis. The T3SS is an essential virulence determinant for many pathogens, but we still lack the structural understanding needed to determine the mechanisms that underlie type III secretion.

NIH Research Projects · FY 2026 · 2022-07

Project Summary Diabetes mellitus is a chronic disorder characterized by insulin deficiency, hyperglycemia and high risk for development of complications of the eyes, kidneys, peripheral nerves, heart and blood vessels. The disease is highly prevalent, affecting more than 30 million people in the U.S. Monitoring of glycemic control has traditionally been considered a cornerstone of diabetes care. The importance of monitoring glycemia has been established by studies proving a direct relationship between mean blood glucose and the development and progression of the chronic complications of diabetes. The landmark Diabetes Control and Complications Trial (DCCT), completed in 1993, showed that risks for development and/or progression of the chronic complications of type 1 diabetes is closely related to the degree of glycemic control, as assessed by HbA1c determinations. The importance of HbA1c as a marker of glycemic control in diabetes mellitus, and more recently as a diagnostic tool, is now well established. This proposed project will improve the measurement of HbA1c for optimal clinical and diagnostic use. This will be done through an established standardization program by continuously tightening criteria for method and laboratory certification, monitoring performance of laboratory methods and laboratories, and testing and publishing data on HbA1c analytical and biological interferences. Preservation of beta cell function in type 1 diabetes has been identified as an important goal in delaying progression of the disease at onset and also for therapeutic intervention. Accurate, standardized measurement of C-peptide will aid in this goal and is also the subject of this proposal. Standardization will be accomplished by implementation of a traceability scheme with re-calibration of C- peptide methods by each manufacturer and monitoring the effects of re-calibration by evaluation of proficiency testing data. In addition, criteria will be formulated for acceptable accuracy, precision and specificity of C-peptide assays.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality in both men and women. Despite rapid therapeutic advances, the 5-year survival rate of EGFR-mutant NSCLC remains a dismal 16% for the past several decades. In particular, the rapid emergence of resistance to widely used treatments, such as the tyrosine kinase inhibitor (TKI), is the key obstacle to achieving long-term NSCLC patient survival. Further, patients with EGFR mutated tumors fail to respond to immune checkpoint inhibitors (ICIs), leaving them with little to no hope of a long-term remission. Therefore, identifying the molecular determinants of resistance and developing a therapeutic that targets the chemo- and immune-resistant pathways, is the best chance to improve survival rates in NSCLC patients with an EGFR mutation. We hypothesize that MU-CN29, the lead targeted siRNA nanoconjugate, restores sensitivity to tyrosine kinase inhibitors in resistant NSCLC. We further postulate that MU-CN29 will induce a systemic immune response, priming the tumor microenvironment for immunotherapy via ICIs. Therefore, the central objective of this TTNCI proposal is to develop, evaluate, and validate MU-CN29 as an effective therapeutic agent for resistant NSCLC. Our promising in vitro and in vivo experimental results confirmed the crosstalk between the resistance-driving receptor tyrosine kinase AXL and FN14 pathways in drug resistant NSCLC. Additionally, we found that the co-knockdown of both AXL and FN14, using MU-CN29, covalently attached with the dual siRNAs, sensitized the resistant tumors to tyrosine kinase inhibitor (TKI), in vitro and in vivo. To enable translation of MU-CN29 to human trials, in this proposal, we will manufacture the nanoparticles as per FDA guidelines, perform detailed toxicology studies in murine and canine subjects, and evaluate the therapeutic efficacy in clinically relevant animal models. The specific aims of this applications are: (1) Determine MU-CN29 clinical-grade production protocols; (2) Determine the safety and efficacy of MU-CN29 in murine NSCLC models; (3) Establish safety of MU-CN29 in canine cancer patients. The data will validate MU-CN29 nanoparticle platform as a promising strategy to combat drug resistance in NSCLC and catalyze clinical trials in the future.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is a global epidemic, progresses to nonalcoholic steatohepatitis (NASH) and fibrosis, and results in the development of hepatocellular carcinoma and increased cardiovascular mortality. Unfortunately, no pharmacological therapies are available yet to treat NASH and fibrosis, necessitating the identification of novel targets and approaches. RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a unique membrane-anchored protein, has been shown to inhibit multiple mediators involved in inflammation and fibrosis. Our novel preliminary data demonstrate that hepatic RECK protein levels are markedly reduced with increasing severity of NASH and fibrosis in clinical patients and in our pre-clinical mouse model of western diet (WD)-induced NASH and fibrosis. Since RECK gene deletion is embryonically lethal, we generated RECK floxed (RECKfl/fl) and CAG-CATflox-RECK transgenic mice. Our proof-of-concept pilot studies demonstrate that while hepatocyte-specific RECK knockdown by AAV8-mediated Cre recombinase exacerbates NASH and fibrosis in short-term WD-fed mice, its overexpression in hepatocytes blunts liver inflammation, Kupffer cells (KC) and hepatic stellate cell (HSC) activation. Moreover, our preliminary in vitro data in primary mouse hepatocytes, KCs and HSCs in which RECK is either silenced or overexpressed support our in vivo studies. These preliminary studies suggest that sustaining RECK expression is hepatoprotective. Therefore, we hypothesize that Cre-Lox mediated RECK deletion specifically in hepatocytes enhances pro-inflammatory signaling by enhancing amphiregulin (AREG) cleavage by ADAM (A Disintegrin And Metalloproteinase domain-containing protein) 10- and 17, leading to increased epidermal growth factor receptor (EGFR) and HSC activation collectively contributing to worsening of long-term WD-induced NASH and fibrosis (Aim 1). Conversely, transgenic overexpression of RECK, specifically in hepatocytes, will be protective. We will also determine if rescuing RECK expression by ectopic overexpression in hepatocyte-specific RECK deficient mice with established WD-induced NASH and fibrosis can be reversed (Aim 1). Since KCs are the predominant resident liver macrophages and HSCs are considered the principal cell type responsible for hepatic fibrosis, we will establish the importance of RECK deletion and transgenic overexpression in Kupffer cells and HSCs on cellular injury and activation, extracellular matrix deposition, and fibrosis (Aim 2). In both Aims, molecular mechanisms underlying inflammation and fibrosis will be investigated in co-culture studies using primary hepatocytes, KCs and HSCs isolated from these gene-altered mouse models. Thus, our proposed genetic and interventional approaches will mechanistically establish RECK as a novel upstream regulator in the pathogenesis of both NASH and fibrosis with potential as a future therapeutic.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY: Pulmonary T cells are critical for host protection from influenza A virus (IAV) infections. While current understanding of influenza immunity is focused on conventional MHC-restricted T cells that recognize peptide antigens, unconventional innate-like T cell subsets, such as CD1d-restricted invariant natural killer T (NKT) cells, are emerging as integral components of the respiratory immune system where they play both a protective and immunopathological role in lung disease. This is principally through their production of large amounts of cytokines in barrier organs like the lung where NKT cells preferentially accumulate. Although NKT cell activities are thought to make up a substantial portion of influenza immunity, relatively little is known about their impact on human infections due to a lack of suitable animal models. The current proposal seeks to address this knowledge gap using swine, which offer an excellent translational model to determine the role NKT cells play in shaping human influenza immunity. Using our extensive expertise in porcine NKT cells and the swine influenza challenge model, we propose three aims: Aim 1 will examine the significance of NKT cells as an important host factor contributing to IAV susceptibility. We have already created NKT cell-deficient CD1d knockout (KO) pigs and discovered that they shed less virus than NKT cell-intact pigs. Aim 2 will use the same pig stock to determine how NKT cells shape the pre-existing immunity afforded by inactivated and modified live virus vaccines. Addressing this question is important to inform vaccinologists since NKT cells have previously been found to generate immune responses that could stimulate durable protection against viral infections. Aim 3 will use our CD1d-KO pigs to determine whether NKT cells modulate vaccine associated enhanced respiratory disease (VAERD), which is a dangerous condition caused by the use of inactivated IAV vaccines containing a virus of the same hemagglutinin subtype as the subsequent challenge strain, but with substantial antigenic shift. This induces non-neutralizing IgG Abs that enhances virus uptake into the target cells. These independent but interconnected aims strongly align with the goals of this funding opportunity: to develop novel models that will accurately reflect influenza immunity in humans, including to better mimic pre-existing immunity for vaccine responses and to better understand special high-risk human populations. Our findings should be of considerable value for reducing influenza transmission and improving vaccine safety and efficiency as there are several therapeutic options to modulate the frequency and function of NKT cells.

NIH Research Projects · FY 2025 · 2022-04

Pathogenesis of coronary artery disease is complex, with multiple cell types contributing to lesion size and composition. Acute coronary syndromes are most often associated with rupture of complex, vulnerable plaques that are otherwise clinically benign. The progression to either a relatively benign, stable lesion or a rupture-prone, vulnerable plaque has been linked to key lesion characteristics, i.e. smooth muscle (SM) and collagen content, macrophage infiltration and necrotic core area within the lesion. The objectives of this proposal are to 1) determine the SM-specific role and underlying mechanism(s) by which the intermediate conductance, Ca2+-activated K+ channel, KCa3.1 (encoded by Kcnn4), dictates atherosclerotic lesion formation and composition and 2) determine the translational potential of clinically approved KCa3.1 inhibitors on lesion development in a large mammal model of coronary artery disease (CAD). In support, we provide the first genetic evidence of a causal link between KCa3.1 and lesion size and SM and macrophage recruitment. The overall hypothesis is that KCa3.1 activation increases migration of SM and macrophages into the intima and contributes to lesion formation. Conversely, blocking KCa3.1, both by genetic silencing or pharmacologically, will decrease atherosclerotic lesion size and beneficially alter composition. Aim 1 will determine the contribution of KCa3.1 in smooth muscle to atherosclerotic lesion formation and composition. Specifically, we will use SM-specific, inducible KO mice to examine the role of KCa3.1 in determining plaque size, composition and gene expression. Aim 2 will define both upstream (REST) and downstream (DOCK2) mechanisms determining KCa3.1 effects on SM and atherosclerosis. We will use genetically modified mice to examine the role of REST and DOCK2 in mediating SM effects of KCa3.1 on phenotype, proliferation, migration, plaque size and composition. In addition, we will use RNA sequencing to identify novel mechanisms of atherosclerosis development by KCa3.1. We will use VSM lineage-tracking in Aim 3 will use SM lineage-tracking to determine role of SMC-KCa3.1 in mediating SMC intimal to medial migration and foam cell transdifferentiation during atherosclerotic lesion development. Finally, Aim 4 will determine the effect of the FDA approved KCa3.1 inhibitor, senicapoc, on atherosclerosis development in a swine model of CAD. We longitudinally track coronary artery disease progression using angiography and IVUS in familial hypercholesterolemic (FH) swine to test the ability of KCa3.1 inhibition with senicapoc, to decrease the size and promote a more favorable composition of coronary lesions. The long-term goal is to provide the pre-clinical foundation for translating current therapeutic tools and developing the next generation drugs targeting KCa3.1 and/or downstream signaling to beneficially manipulate atherosclerotic lesion composition.

NIH Research Projects · FY 2025 · 2022-04

Effect of Hearing Aid Insurance Coverage Requirements for Adults on Utilization Abstract Hearing loss affects 23% of those aged 12 and older in the U.S. More than 2/3 of U.S. adults over the age of 70 have significant hearing loss. The most efficacious management option for most individuals with hearing loss is the use of hearing aids (i.e., a small removable electronic device that is worn in or behind the ear to amplify sound), which can improve communication and quality of life. Hearing aids are generally not covered by insurance and about 86% of hearing loss cases go untreated. However, Medicaid provides hearing aid benefits in 28 states and 8 states require private insurance benefits for at least some adults. These coverage requirements may improve hearing aid uptake by reducing costs as a barrier. The long-term goal of our research agenda is to understand the effects of improving hearing on health. Specifically, the objective of our current application is to use quasi-experimental (e.g., difference-in-differences) evaluation methods to estimate the effect of coverage requirements in Medicaid and private insurance plans on hearing aid use. We will use high quality, reproducible Anthem private insurance claims data, National Health Interview Survey (NHIS) data, Medicaid claims-linked NHIS data, and Medicare Current Beneficiary Survey (MCBS) data to study: (i) the effect of state private insurance coverage requirements for hearing aids on beneficiary hearing aid purchasing and out-of-pocket payments in fully-insured health insurance plans; (ii) the effect of Medicaid and private insurance coverage requirements on use of hearing aids in the NHIS, and Medicaid coverage requirements in the Medicaid-linked NHIS and MCBS (for dual-eligibles); and (iii) the effect of private and Medicaid requirements by demographic group. Our project is significant by focusing on high rates of untreated hearing loss; by estimating the effects of state implementation of private and public insurance coverage requirements on hearing aid adoption among U.S. adults (which provides evidence for the effects of pending national legislation); and by providing evidence on whether hearing aid coverage requirements improve well- documented disparities in hearing aid use for males, racial/ethnic minorities, and lower-educated individuals. Our project is innovative by being the first to use quasi-experimental difference-in-differences methods (which can provide causal evidence) for hearing health care research, including newer methods accounting for heterogeneous treatment effects; leveraging powerful sources of currently under-used data to study hearing health care access and utilization; constructing detailed coverage requirement data through original policy research that we will make available publicly. The research team includes experts in clinical audiology, health economics, advanced statistical modeling, insurance claims data, and health policy. The proposed project will have a positive impact because addressing system barriers to affordable and accessible hearing health care has the potential to reduce disparities and increase use.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT: Current understanding of cardiac parasympathetic (or vagal) activity unequivocally demonstrates that the vagal activity to the heart and homeostatic reflex changes in cardiac vagal activity are mediated by cardiac vagal motor neurons (CVNs) in the brainstem. Therefore, CVNs play an essential role in normal cardiovascular function. Despite our understanding of CVN neurophysiology in health, the potential for CVN dysfunction in diseases is still unclear. This is particularly true of our understanding of the complex interplay between diet and cardiovascular function. In some estimates, Americans are consuming 600 more calories from fat per day then any time in the recent past. This increased consumption of foods high in fat significantly elevated the risk of developing cardiovascular diseases. A distinctive hallmark of cardiovascular disease risk is low cardiac vagal signaling, and the extend of this imbalance correlates strongly with increasing risk morbidity and mortality. Preliminary data from our laboratory demonstrate that CVN activity is significantly reduced and inhibitory neurotransmission to CVNs is increased during early consumption of foods high in fat. This reduced CVN activity parallels a significant reduction in cardiac vagal contribution to resting heart rate. The reduction in vagal activity can be abolished through genetic knock down of a specific subunit of the receptors that mediate inhibition in CVNs. Critically, PKC inhibition also abolishes the influence of high fat diet on vagal function, and this increased PKCδ activity is likely mediated by increased activity of the alpha-1 adrenoreceptor on CVNs. Our overall hypothesis guiding this proposal is that high fat diet-induced increase in functional expression of inhibitory receptors in CVNs results in a progressive decline in overall vagal activity. However, critical questions remain, including how quickly does the increased inhibition of CVNs occur and what role does the PKCδ isoform play in this inhibition. Therefore, this proposal will 1) quantitively determine the timing of cardiac vagal signaling after high fat diet, and 2) establish the role of PKCδ in the effects of early HFD on CVN function. The anticipated results of these experiments will provide fundamental details in our understanding of cardiac vagal regulation and mechanisms responsible for vagal regulation of heart rate. Identifying the early mechanistic consequences of HFD on vagal activity could lead to the discovery of biomarkers, and early testing of new therapeutics targeting disease mechanisms, rather than symptoms.

NIH Research Projects · FY 2024 · 2022-03

PROJECT SUMMARY/ABSTRACT Autism Spectrum Disorders (autism) has significant and long term effects on the lives of children and their families. Reliable diagnosis of autism is not possible until 2 years of age or later, and there are no currently available methods to screen for autism in early infancy. Preliminary evidence suggests that children with autism may be characterized by atypical features of cry and neurobehavior during early infancy. Previous research has identified atypicalities in vocal production in older children with autism, and unusual acoustic features of infant cry would be consistent with such findings from early childhood. One barrier to the acoustic analysis of newborn cry has been the lack of a computerized cry analysis system based on modern technology. Our lab has developed a new cry analysis system based on state-of-the-art signal processing that can be used to study acoustic characteristics of cry that could relate to risk for autism. Our group has also developed the NNNS, a widely used and well validated neurobehavioral exam for infants in the newborn period. Preliminary findings point to a set of cry and neurobehavioral features from these measures that was able to differentiate a sample of infants later diagnosed with autism from non-autistic infants included in a longitudinal, heterogeneous, child development study. Other preliminary findings support the hypothesis that infant cry and neurobehavior are affected in infants with later autism diagnoses. In the proposed project, we will utilize cry and neurobehavioral assessments that are being collected as part of unique pregnancy and birth cohort at a large regional hospital. It is anticipated that up to up to 5,000 infants will be enrolled and followed longitudinally. A 2-stage screening and evaluation process will identify children with autism by 24 to 36 months of age. Ongoing analyses will be conducted to identify neonatal cry and neurobehavioral characteristics that are associated with risk for autism using signal detection methods and complex conditional statistical models. This project is of high significance and has the potential to have substantial impact on public health by identifying potential indicators of risk for autism. The long term impact of this research would be on the development of early screening and early interventions for infants at risk for autism.

NIH Research Projects · FY 2025 · 2022-03

SUMMARY In the study of disease, the polygenic factors that lead to genetic adaptation in species, essentially who will be asymptomatic while under environmental stressors, mostly remain undiscovered. Without a comparative understanding of these unique features by cell type, that evolution has preserved, our efforts to more broadly implement precision medicine will be limited in multiple phenotypes. One of the most promising applications of evolutionary medicine has been the use of divergent animal models to deconstruct and uncover fundamental concepts of biological organization in great detail. The Mexican cavefish, Astyanax mexicanus provides a rapidly growing model system to apply principles of evolutionary medicine to study trait variation. We have implemented a toolbox of genetic tools that allow for functional interrogation of various traits in the Mexican cavefish, but key resources are missing. This model species consists of surface and cavefish populations that possess natural trait differentiation, often displaying genetic adaption associated with environmental change, without impacting health or longevity. The objectives of our studies are to generate three tiers of community requested resources: high-quality surface and cavefish genome assemblies, using powerful single-cell sequencing technology a cell atlas with differentiating gene expression data sets and targeted gene reporter constructs coupled with a spatial understanding of their gene regulatory effects. This compendium of resource data and methodology can serve a large community wishing to test various hypotheses of adaptation in this species and others. Our proposed research objectives are significant in that they will contribute comparative gene networks that reveal novel differences with a growing assemblage of gene candidates for human diseases.

NIH Research Projects · FY 2026 · 2022-03

Linking Science, Mathematics and Literacy (LSM&L) ABSTRACT The proposed program will provide innovative educational resources to help redress significant nationwide deficits in science, mathematics, and literacy learning, especially for grades 6-8 students with disabilities (SWD). These new resources will improve and extend the: 1) multimodal STEM text sets: 2) virtual teacher professional development – communities of practice (vPD-CoP); and 3) scenario-based assessments (SBAs) of scientific argumentation developed by the current NIGMS/SEPA Linking Science and Literacy for Learners program, and that have garnered state and national recognition. Multimodal STEM text sets (1) are coherent collections of content and instructional resources pertaining to an anchor phenomenon and line of inquiry that support learners’: a) Engagement with a grade-band level complex anchor text; b) Development of the science and disciplinary literacy skills called for by the shared Next Generation Science Standards (NGSS) and Common Core State Standards (CCSS) or state equivalents. The vPD-CoPs (2) help teachers, learn about, develop, and implement multimodal STEM text sets for their grade 6-8 learners. The SBAs (3) provide quantitative data about the instructional interventions and help teachers understand scientific argumentation practices and include them in their instruction. Preliminary evidence supports the hypothesis that appropriate instructional support with multimodal STEM text sets, helps students (particularly SWDs) engage with grade-band level complex anchor texts and improve scientific argumentation skills that can be measured with SBAs. The proposed program will add mathematics and research-generated data to all the program resources, and the effects will be rigorously tested over five years with quantitative data from thousands of students nationwide, and with qualitative data of instructional implementation by teachers of science, mathematics, English language arts and special education in school settings, nationwide. These resources and the research data will be disseminated widely by the program website, in high profile journals, in books and reports, and at regional and national meetings, to have a lasting impact upon students’ participation in the biomedical workforce and contributions to society. The program faculty and consultants are experienced educator/researchers in science, literacy, mathematics and special education with a strong independent evaluation team and institutional support.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Nicotinamide adenine dinucleotide (NAD+) is a cofactor required for glycolysis, the tricarboxylic acid cycle (TCA) and enzymatic reaction in electron transport chain (ETC). In mammalian cells, NAD+ salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme, is the predominant pathway for NAD+ biosynthesis. Although the dysregulation of NAD+ in aging and neurodegerative diseases has been reported, genetic diseases caused by NAMPT variants have not been clinically recognized and understood. Here we identified the first case of an inherited neurological disease caused by a homozygous single nucleotide polymorphism (SNP), i.e., a P158A mutation in the coding region of NAMPT gene. The major clinical features of patients include impaired motor coordination, muscle weakness, atrophy of lower extremities, positive Babiński sign. The patients were diagnosed as hereditary motor and sensory neuropathy involving axonal degeneration and neuromuscular junction (NMJ) dysfunction. Using skin-derived patient fibroblasts (p-FBs), our preliminary studies found that P158A mutation causes reduced bioenergetics, mitochondrial dysfunction, and decreased enzymatic activity of NAMPT for NAD+ biosynthesis compared with healthy control fibroblasts (c-FBs). The results indicate the pathological conditions related to the patients is initially resulted from bioenergetic stress and ultimately from neuronal and muscular degeneration. Thus, our project goal is to understand the pathogenesis and the mechanism of neuronal and muscular degeneration of this new disease. To achieve our goal, we generated many molecular tools including P158A-NAMPT mutant mice, c- & p-FBs-derived induced pluripotent stem cells (c- & p-iPSCs including isogenic and patient like p-iPSCs), and iPSC-induced motor neurons (c- & p- iMNs). We propose three Specific Aims. Aim 1 will test the hypothesis P158A mutation in NAMPT causes mitochondrial and synaptic dysfunction of p-iMNs. Using iMNs, we will study the effect of P158A mutation on cellular bioenergetics, glycolytic metabolism and mitochondrial respiration. We will also conduct combined metabolomic and transcriptional profiling to determine the molecular base of metabolic changes caused by P158A mutation. Aim 2 will test the hypothesis that P158A mutation in NAMPT causes MN degeneration. Using the mutant mice, we will study disease progression, upper and lower MN degeneration. We will use electrophysiological and two-photon (2-P) imaging to study the effect of P158A mutation on sensory response and cytosolic and mitochondrial Ca2+ signaling. Aim 3 will test the hypothesis that P158A mutation in NAMPT causes NMJ abnormalities and muscle degeneration. We will assess structural and functional abnormalities of NMJs and muscle contractile response of semitendinosus muscles isolated from the symptomatic mutant mice. A human disease caused by NAMPT mutation has not been reported so far. Our application represents a first in-depth study on the pathogenesis and mechanism of motor neuron and muscle degeneration of a new neurological disease caused by a mutation in NAMPT gene.

NIH Research Projects · FY 2023 · 2021-12

Project Summary Hearing loss is the third most common health condition, affecting people of all ages. For individuals who are deaf or have significant hearing loss, cochlear implants (CIs) are a standard treatment. CIs restore hearing by electrically stimulating residual viable auditory nerve fibers in the cochlea with an implanted electrode array. However, cochlear implantation is always accompanied by surgical injury, which initiates an acute inflammatory response to the electrode and induces on-set and progressive loss of residual acoustic hearing. Thus, there is an urgent need to develop a real-time monitoring method to help understand the neural conditions during and after implantation and subsequent hearing loss to enhance post-operative clinical outcomes. Inflammatory process induces oxidative stress (i.e. an elevated intracellular level of reactive oxygen species (ROS)) and reduces cellular antioxidant capacity. Numerous studies have shown that oxidative stress plays key roles for chronic neuroinflammation. Therefore, we hypothesize that oxidative stress is the major factor compromising CIs’ clinical outcome. In this R21 project, we propose to develop novel multifunctional CI electrodes and methods that can simultaneously monitor and scavenge initiation of the oxidative stress pathway during and after cochlear implantation. We will develop the multifunctional CIs to achieve real-time in vivo sensing and scavenging of ROS with clinically required sensitivity and specificity. Our approach to develop advanced multifunctional CIs is to utilize the unique palladium (Pd) and Pd bimetallic (i.e. Pd/Au) nanoparticles that show enzyme-like activities allowing sensitive and selective sensing of ROS as well as converting ROS to neutral molecules. In contrast to existing surface technologies for auditory neuronal protection and regeneration, our noble metal nanocatalysts as CIs electrodes are corrosion-resistant and biocompatible, and their unique surface electrochemistry can provide continuous (during and post-operative times) sensing and removal of ROS in vivo with high stability. The simultaneous neutralization of ROS with their electrochemical sensing reactions should mitigate oxidative stress-related cell death without disrupting the well- integrated innate antioxidant defense network, thus, improving post-operative clinical outcomes for individuals with CIs. In Aim 1, we will design and fabricate multifunctional CIs with bimetallic Pd/Au nanocatalysts integrated into a flexible parylene-based electrode array for detecting and scavenging ROS in vitro. In Aim 2, we will validate the detection and scavenging functions of the multifunctional CIs with an integrated microchannel in a rat model. Results from these pre-clinical development and validation studies in this R21 project will form the basis of a long-term R01 project to fully integrate sensing/scavenging capability into CI devices that are capable of recording, stimulation, sensing and scavenging functions for long-term clinical use. We also foresee the opportunities to apply the results gained here to other neural interfaces and medical implants. Our established multidisciplinary team with each member has more than 20 years’ experience with in vivo neuroprobe, real-time chemical sensor technology, and auditory neurophysiology respectively, making us well prepared to be successful in the proposed research activities.

NIH Research Projects · FY 2025 · 2021-12

The dedifferentiation of vascular smooth muscle cells (SMCs) into synthetic SMCs, a hallmark of many occlusive vascular diseases, is associated with a metabolic switch that is characterized by increased aerobic glycolysis, which also fuels mevalonate metabolism, decreased glucose oxidation and increased fatty acid oxidation. However, the molecular links between environmental cues and the metabolic reprogramming remain poorly understood. Our pilot studies revealed that cyclin dependent kinase 8 (CDK8) is a master regulator of the metabolic control of vascular SMC dedifferentiation for intimal hyperplasia toward vascular occlusion. Mechanistic investigations uncovered that CDK8 controls the SREBP2 (sterol regulatory element binding factor-2)-operated transcription to promote the mevalonate metabolism for protein geranylgeranylation, which drives the vascular SMC dedifferentiation. Thus, we propose a novel paradigm in which CDK8 controls the mevalonate metabolism for protein geranylgeranylation to promote the dedifferentiation of vascular SMCs for intimal hyperplasia, thereby contributing to occlusive vascular disease. We will test this hypothesis and delineate the molecular mechanisms of CDK8-operated metabolic control of vascular SMC dedifferentiation by 2 specific aims: Aim 1 will establish a mediator role of CDK8 in vascular SMC dedifferentiation into synthetic SMCs for intimal hyperplasia toward vascular occlusion; Aim 2 will determine the underlying molecular mechanisms with a focus on the molecular network by which CDK8 operates the mevalonate metabolism pathway for protein geranylgeranylation which is required for vascular SMC dedifferentiation into synthetic SMCs leading to intimal hyperplasia toward vascular occlusion. This proposal will provide the first assessment of CDK8-mediated occlusive vascular lesion formation and define a novel pathway of occlusive vascular remodeling that is mediated by previously unrecognized CDK8-operated metabolic reprogramming for vascular SMC dedifferentiation, thus shedding light on the study of vascular SMC plasticity as well as the development of innovative and effective therapeutic approaches for occlusive vascular disease.

NIH Research Projects · FY 2025 · 2021-09

Copper (Cu) is an essential nutrient that plays vital roles in oxygen transport and utilization. Copper functions directly in the consumption of oxygen via oxidative phosphorylation and is required for the transport of iron which is a vital for oxygen transport within hemoglobin. Despite the importance of copper in oxygen metabolism, little is known regarding the effects of reduced oxygen levels (hypoxia) on copper homeostasis. Hypoxia is a physiological stress that contributes to the pathology of many common diseases, thus the mechanisms by which cells sense and respond to hypoxia is of fundamental importance to human health. Using an innovative CRISPR-based knockout screen for novel regulators of copper homeostasis, mutations in the von Hippel Lindau (VHL) gene were found to stimulate the expression of ATP7B, a copper transporter primarily expressed in hepatocytes. VHL is a master regulator of oxygen sensing and we demonstrate that ATP7B expression is strongly induced by hypoxia in cultured cells and in the liver of mice. ATP7B is essential for inserting copper into the ceruloplasmin, a ferroxidase with known roles in iron export into the plasma. In this proposal, we will test the hypothesis that ATP7B is required for hypoxia-induced erythropoiesis through its ability to facilitate ceruloplasmin-mediated iron export into the plasma. Mutations in the ATP7B gene are known to cause Wilson disease, a lethal disorder of hepatic copper overload. We demonstrate that hypoxia induces hepatic expression of an alternative copper transporter, ATP7A, which is a functional homologue of ATP7B. In this proposal, we will test the hypothesis that hypoxia-induced ATP7A can attenuate hepatic copper overload and liver pathology in the ATP7B-/- mouse model of Wilson disease. To increase the translational potential of our studies, the therapeutic efficacy of a clinically approved hypoxia-mimetic drug Roxadustat will also be tested in ATP7B-/- mice to address its potential for repurposing as a novel treatment for Wilson disease. Finally, based on the demonstrated success of our knockout screen, we will perform an innovative CRISPR- based gene activation screen for novel regulators of copper homeostasis. To test these hypotheses, our proposal will 1) investigate the roles of copper in adaptive responses to hypoxia; 2) test the therapeutic potential of hypoxia in animal models of Wilson disease and 3) identify novel components of mammalian copper homeostasis using an innovative gene activation screen.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT: Primary open angle glaucoma (POAG), the most common form of glaucoma, is characterized by progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible vision loss. Elevated intraocular pressure (IOP) is the major risk factor for POAG. Unfortunately, the underlying pathological mechanisms responsible for IOP-induced glaucomatous neurodegeneration still remain unclear. The long-term goal of this proposal is to delineate the molecular pathways governing IOP-induced glaucomatous neurodegeneration and to develop an effective glaucoma treatment strategy. To this end, we have developed a novel glucocorticoid (GC)-induced and myocilin-associated mouse models of POAG, replicating human POAG phenotypes. Importantly, we have identified impaired mitophagy, accumulation of damaged mitochondria and inflammatory immune cells in the optic nerve of both human and mouse glaucoma. Based upon our preliminary data, we propose to: 1) examine the effect of IOP on mitophagy impairment and accumulation of damaged mitochondria using mouse and human POAG, 2) examine the role of impaired mitophagy on glaucomatous neurodegeneration and, 3) further identify whether enhancing mitophagy alleviates neurodegeneration and prevent RGC loss in mouse models of POAG. In the mentored phase, I will establish mouse models to study mitophagy in POAG including mitophagy reporter transgenic Mt-Keima mice, RGC-specific Parkin and ATG5 conditional knockout mice under the guidance of Dr. Gulab Zode (an expert in chronic ER stress and autophagy in trabecular meshwork). In collaboration with Dr. Denise Inman (an expert in the field of mitochondrial metabolism), I will enhance my understanding of mitochondrial dysfunction in mouse models of POAG. The mentored phase will also be supplemented by training with Dr. Abbot Clark (well-known leading glaucoma expert), who will provide assistance with human tissues as well as an additional training for my independent career. Furthermore, regular meetings with Dr. Paula Gregory who has tremendous experience in assisting young investigators will help me to develop independent career. During the independent phase, we will examine role of mitophagy on inflammatory neurodegeneration and determine whether inducing mitophagy (Urolithin A/Actinonin/Metformin/ Parkin overexpression) rescues GC or myocilin-associated POAG. Additionally, during the mentored phase, I will be having regular meetings with my advisory committee, attend scientific conferences, and continue my career development. I am in the ideal environment for the proposed research and for my career development as Dr. Zode has an established state-of-the-art facilities at the North Texas Eye Research Institute, and collaborations with renowned scientists, Dr. Val Sheffield, Dr. John Hulleman and Dr. Kevin Park. This will help me to set-up good collaborations, learn new techniques, and build an independent research laboratory at a well- established academic institution.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The fact that both duplication and deletion of the Peripheral Myelin Protein 22 (PMP22) gene cause dysmyelinating peripheral neuropathy illustrates the importance of PMP22 for peripheral myelin integrity. PMP22 duplication causes Charcot-Marie-Tooth Disease Type 1A (CMT1A) and PMP22 deletion causes Hereditary Neuropathy with Liability to Pressure Palsies (HNPP). Although CMT1A and HNPP are the most common inherited peripheral neuropathies, research on them is underfunded and there are currently no disease-modifying treatments. This is largely due to the fact that PMP22 function and the consequence of altered PMP22 expression remain unclear. Thus, there is a critical need to expand knowledge of what PMP22 does in myelin, how it is regulated and when precise expression is required as a means to improve therapeutic potential of CMT1A and HNPP. This proposal aims to utilize cutting-edge techniques, including conditional mouse models and super resolution microscopy, and knowledge of cell adhesion and membrane biophysics to advance understanding of CMT1A and HNPP pathomechanisms. My central hypothesis is that PMP22 gene dosage and lipid raft association govern localization and organization of myelin adherens and tight junctions; a function that is most critical during development. In Aim 1, I will determine how PMP22 regulates adhesion junction organization in peripheral nerve myelin during development and aging with super resolution and electron microscopy. I will aid the interpretation of these studies by evaluating the effects of altered PMP22 expression on prototypical adherens and tight junctions in Madin-Darby Canine Kidney (MDCK) epithelial cells. The temporal requirement for precise PMP22 expression in myelin will also be defined by generating powerful conditional mouse models of CMT1A and HNPP. In Aim 2, I will determine how palmitoylation impacts PMP22 lipid raft association and regulation of adhesion junctions and define the biophysical properties of PMP22 within the plasma membrane using MDCK and Schwann cell models of CMT1A and HNPP and advanced biophysical methods. This K22 Career Transition Award will provide me with additional training, mentorship and expertise in cell adhesion, membrane biophysics and microscopy, thereby enabling my proposed research and facilitating my transition to independence. This training will complement my previous training in cell and molecular neurobiology and my current peripheral nerve training, and the expertise acquired during Phase I will be applied to more complex models in Phase II to expand mechanistic details. Completion of these aims will accelerate progress towards my long-term goal of developing an independent academic research career studying pathomechanisms of CMT1A and HNPP as a means to improve their therapeutic potential. The training and mentorship provided by this award will expand my technical skills and expertise, positioning me for success as an independent investigator.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY The overall goal of this proposal is to find novel mechanisms whereby surfactant protein A (SPA) regulates vascular smooth muscle cell (SMC) phenotype modulation. SMC transition from a differentiated to dedifferentiated phenotype in addition to neointima formation/vascular remodeling has a critical role in human diseases such as the development of atherosclerosis, restenosis after angioplasty or bypass, diabetic vascular complications, arteriopathy transplants, asthma and cancer. Mechanisms that regulate SMC phenotype modulation and neointima formation are not well understood. The physiological function of SPA is its secretion by type II alveolar cells to maintain minimal surface tension in the lungs. However, preliminary data indicate a role for SPA as a SMC phenotype modulator. In vivo, SPA was expressed in the medial and neointimal SMCs following mechanical injury in rat and mouse carotid arteries. The wire-injury induced intimal hyperplasia was dramatically attenuated in SPA knockout mice. Furthermore, increased mRNA expression of SMC contractile genes and key regulators for contractile SMC phenotype, Myocardin and TGF-β1 was observed in SMCs isolated from SPA knockout mice. Additionally, SMCs from SPA knockout mice had increased Smad3 phosphorylation and the increase was blocked by the TGF-β1 neutralizing antibody. SPA is localized in the nucleus of SMCs suggesting it may have a role in SMC gene transcription. Indeed, SPA deficiency increased smooth muscle α-actin and smooth muscle 22-α promoter activity whereas recombinant SPA protein attenuated their activities. Hence, the central hypothesis is that SPA regulates SMC phenotype modulation and vascular remodeling through both extracellular (via modulating TGF-β1 signaling) and intracellular (Myocardin-related gene transcription) mechanisms. Using primary culture of SMC, in vivo mouse wire injury models combined with molecular, cellular and histological approaches, this proposal will 1) determine the molecular extracellular and intracellular mechanisms by which SPA regulates SMC phenotypic modulation; and 2) determine if SPA is essential for SMC phenotype modulation/vascular remodeling in vivo. Project completion will uncover novel mechanisms regulating SMC phenotypic modulation and provide understanding into whether SPA is a potential target for therapy against vascular damage associated with common vascular diseases such as diabetes, restenosis, atherosclerosis and cancer. The training plan laid out by the sponsor and the outstanding environment in the mentor’s laboratory and at the University of Missouri will safeguard the successful completion of the proposed studies.

NIH Research Projects · FY 2025 · 2021-09

Project summary Meiosis is a specialized set of cell divisions that produce haploid gametes. During meiosis I (MI) in females, bipolar spindle formation and positioning within the oocyte must be regulated tightly to ensure faithful chromosome segregation and proper genome inheritance. In somatic mitotic cells, bipolar spindle formation and positioning rely on a centrosome pair, each of which contains two centrioles. Interestingly, meiotic oocytes lack centrioles and, hence, lack classic centrosomes. Meiotic oocytes, instead, contain numerous microtubule (MT) organizing centers (MTOCs) that are organized, by largely unknown mechanisms, to establish two spindle poles (polar MTOCs). The traditional view was that, in mammalian oocytes, MTs (and their associated proteins) are the only cytoskeletal components responsible for organizing such MTOC spindles. However, recent data suggest that F-actin is also involved in spindle bipolarity regulation. How F-actin interacts with MTs to regulate polar MTOC organization during MI represents a critical gap in our understanding of how the meiotic spindle is built. We recently identified a novel, functionally different, class of MTOCs (mcMTOCs) and found that spindle maintenance at the oocyte center is regulated by two opposing forces (mcMTOC-mediated MTs vs. F-actin). We also recently observed that ~50% of spindles are not assembled centrally. To date, such peripheral spindle assembly was unobservable owing to technical limitations associated with spindle fluorescence (i.e. live imaging). To circumvent this, we generated a Cep192-eGfp reporter mouse model enabling spindle tracking wherever it is assembled. Strikingly, peripheral spindle formation is typically followed by spindle migration towards the center – a previously undocumented phenomenon. Understanding the molecular mechanisms regulating this corrective developmental event represents a major gap in our knowledge of meiotic spindle spatiotemporal regulation during MI. This proposal lays the foundations for our long-term goal: To understand how two critical events during MI — bipolar spindle assembly and positioning — are regulated, in the absence of centrioles, to ensure faithful chromosome segregation. To do so, we will utilize state-of-the-art approaches, including transgenic mouse models, genetic constructs, laser ablation, and cutting-edge imaging, to tackle three critical goals: (i) determine how F-actin interacts with MTs to organize polar MTOCs during bipolar spindle building, (ii) establish the mechanism(s) by which the peripheral acentriolar spindle migrates to the oocyte center, and (iii) determine whether differences in biochemical compositions of mcMTOCs vs. polar MTOCs underlie their functional differences. Given that chromosome segregation errors (very common during MI) lead to aneuploidy, the leading genetic cause of developmental disorders and miscarriage, these studies have the potential to significantly advance our basic understanding of two fundamental processes — spindle formation and positioning — during MI whilst simultaneously shedding light on why MI is notoriously error prone.

NIH Research Projects · FY 2022 · 2021-09

Project Summary: The long-term goal of this project is to develop a paradigm-shifting neurosensing technology for direct, simultaneous monitoring of the activity of multiple neurotransmitters for understanding brain function. The retina is selected as our model system due to its easy accessibility and well-established neurophysiology and the urgent needs in such tool to understand the roles of neurotransmitters in various eye diseases such as diabetic retinopathy. Retinal photosensitive cells (rods and cones) convert light into an electrical signal. The electrical signal is transmitted through bipolar cells to ganglion cells, the output neurons of the retina, and then to the brain. Signal transmission through this pathway is modulated by amacrine cells, which are retinal interneurons. There are multiple types of amacrine cells, but all synthesize and release neuromodulators such as dopamine (DA), gamma-aminobutyric acid (GABA) and acetylcholine (ACh). Specifically, dopaminergic amacrine cells (DACs) co-release GABA and DA, which play a critical role in modulating retinal light sensitivity and eye development. Starburst amacrine cells co-release GABA and ACh, which initiates the motion direction of the visual system. Historically, the release of neurotransmitters from retinal neurons and amacrine cells has been studied indirectly, through electrophysiological methods and/or redox detection using electroanalytical techniques employing carbon fiber microelectrodes. However, electrical activity in a cell does not always match the release of neurotransmitter from the cell. Redox methods only work for a relatively small number of analytes such as DA. We have constructed a novel biosensor that employs complementary electrochemical and piezoelectric sensors, and our preliminary results show that it can differentiate between redox and non-redox active neurotransmitters. The objective of this R21 project is to develop a miniaturized multimodal biosensor to measure multiple neurotransmitters simultaneously with high spatial and temporal resolution in real time, label- and reagent-free with two Aims: 1. Design, fabrication, and characterization of a miniaturized multimodal electrochemical (E) and piezoelectric sensor (thin film bulk acoustic resonator (FBAR) (i.e. E-FBAR) neurosensing probe; and 2: Validation of the neurosensing probe through monitoring dopamine, GABA, and ACh in living normal and diabetic retinal neurons. Successful completion of this project will certify a reagent-free, label-free and real-time simultaneously detection of both redox active and non-redox active neurotransmitters in retina with multifaceted information in high sensitivity and selectivity. Such a tool will be invaluable to research aimed at understanding the causes and mechanisms responsible for retinal neurodegenerative diseases such as diabetic retinopathy, and also to test therapeutic agents for the treatment of such diseases. This novel technology could also be adapted to monitor other important neurotransmitters in the brain, increasing our understanding of brain functions. Our well- established, highly skilled, multidisciplinary team has the expertise in electrochemical and acoustic biosensors, microdevice and microsensor design and fabrication, and visual neuroscience to develop and validate the proposed neurosensing technology.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The overall goal of this project is to investigate the role of Zika virus (ZIKV) in glaucoma pathobiology. ZIKV is an emerging viral pathogen that causes microcephaly and leads to severe ocular complications in newborns born to ZIKV infected mothers. Although the ocular manifestations of ZIKV are primarily reported to affect the posterior segment of the eye resulting in chorioretinal atrophy, withering of the retina and choroid, and optic nerve abnormalities, several clinical case reports showed the involvement of the anterior segment resulting in glaucoma. Studies from our laboratory, as well as those of others, have shown that ZIKV can cause glaucomatous pathology including an increase in intraocular pressure (IOP), retinal ganglion cell (RGC) loss, and optic nerve damage. The offspring of ZIKV infected dams have shown increased IOP and RGC loss and the presence of anti-flavivirus-antibody in these mice correlates with significantly enhanced glaucoma pathology due to antibody-dependent enhancement. Until the recent ZIKV epidemics, glaucoma has been primarily considered as a genetic and age-related disease and has not been reported among infants exposed to infection during gestation. Several studies have now reported that ZIKV can cause congenital glaucoma in infants born from mothers who were infected during pregnancy. Considering the fact that there is an endemic transmission of ZIKV in >84 countries, it is imperative to investigate the link between ZIKV and glaucoma to develop new prognostic and therapeutic tools to combat this global health threat. Our laboratory has developed several in vitro and in vivo models to study the pathobiology of ocular ZIKV infections. In our recent study, we reported that ZIKV can infect and replicate in human primary Trabecular Meshwork cells (HTMC). More recently, we performed RNAseq analysis and discovered that ZIKV infection of HTMC leads to transcriptomic alteration and dysregulation of several pathways including those that modulate ER stress response, autophagy, hypoxia, and ECM organization. Furthermore, ZIKV-infected mice exhibited increased IOP, ER stress, and autophagy in the anterior segment of the eye. ZIKV infection also caused RGC death and loss of RGC and optic nerve damage leading to disruption of anterograde axonal transport. Based on these novel findings, we hypothesize that ZIKV induces ER stress and autophagy resulting in TM death and dysfunction, increased IOP, and the development of glaucoma. Two specific aims are proposed to test this hypothesis. Aim 1 will determine the role of ZIKV induced ER stress in TM dysfunction and the pathobiology of glaucoma using C57BL/6 (WT) and IFNAR1-/- mice/pups and whether the reduction of ER stress alleviates ZIKV induced glaucomatous pathology. Aim 2 will investigate the role of autophagy using HTMC, and mouse models and evaluate the therapeutic efficacy of an FDA approved drug, hydroxychloroquine (HCQ) in ZIKV induced glaucoma. The anticipated results will establish the role of ZIKV infection in the pathogenesis of glaucoma and elucidate the molecular mechanisms and pathway-mediated therapeutic targets for future treatments.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT The COVID-19 pandemic highlighted the vulnerability of the nearly 1.4 million nursing home (NH) residents to respiratory healthcare associated infections (HAI) such as COVID-19. By August 2020, there were nearly 200,000 confirmed cases of COVID-19 with more than 50,000 reported resident deaths. NH residents are vulnerable to respiratory HAI because of multiple comorbidities and physical and cognitive frailty. These vulnerabilities are compounded by an institutional environment of common caregivers, shared living spaces, and a setting with a history of being under-resourced and ill-prepared to manage infection outbreaks. While COVID-19 related NH infections and mortality continue to increase nationwide, no one has reported on the impact of COVID-19 on NHs’ capacity to respond to the pandemic and to understand the impact of their response on clinical, functional, and psychosocial resident outcomes jointly. We are proposing a longitudinal mixed-methods study with the goal to develop knowledge and recommendations to improve US NHs’ ability to respond to respiratory HAI outbreaks. We have unique access to data recorded by the Quality Improvement Program for Missouri (QIPMO), a state-sponsored cooperative program. In March 2020, QIPMO began documenting NH COVID-19 infections and support provided to Missouri NHs. By June, there were over 2,400 documented QIPMO encounters providing us an unprecedented opportunity to study how NHs responded to COVID-19. In aim 1, we will use QIPMO data, state key informant interviews, and interviews with over 300 leaders and staff from 24 purposively sampled NHs to assess diverse NH pandemic responses, including enactment of federal guidance. In aim 2, using an interrupted time-series analysis, we will leverage statistical data from the Minimum Data Set (MDS) to determine the effects of the COVID-19 response on NH resident clinical, functional, and psychosocial outcomes. In aim 3, we will converge findings from aims 1 and 2 to identify relationships between contextual differences in NH responses and resident health outcomes to describe practices and strategies that either mitigated or contributed to adverse outcomes. Finally, in aim 4 we will convene an expert panel to review and recommend updates to current NH HAI guidelines and identify new practices and strategies to enhance NHs’ capacity to respond to infectious disease outbreaks including identifying implementation barriers, final dissemination plans and future intervention development. The likelihood that COVID-19 will become endemic and create needs for ongoing management is growing. Understanding how NHs’ responses influenced outcomes, including long-term resident effects, will inform future intervention development for US NHs to systematically prepare and manage infectious disease outbreaks. This study addresses the AHRQ priority to better prepare NHs for prevention and management, specifically high-risk respiratory HAI illnesses like COVID-19.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY: Tick-transmitted rickettsial diseases of the genera Anaplasma, Ehrlichia, and Rickettsia remain a growing public health concern in the USA and many parts of the world. The diseases include one of the oldest known rickettsial diseases, Rocky Mountain spotted fever (RMSF) caused by Rickettsia rickettsii. RMSF remains a serious disease of people and continues to be a public health concern in the USA and several North, Central and South American countries. Clinical signs of RMSF include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. The disease can progress rapidly to a life-threatening illness in untreated patients, resulting in high mortality rates ranging from 30-80%. During the last two decades, reported RMSF cases continue rising in parts of North America. Tick-borne diseases (TBDs) require the interplay of humans, ticks and reservoir animal hosts. We believe that developing a vaccine to prevent the disease can be accomplished by engaging in collaborative research with a team of experts having diverse expertise and yet having common broad research interests. Since dogs develop disease similar to people, a vaccine to prevent the disease in this host will most likely be effective in controlling the disease spread from wildlife, ticks and also infections from dogs to people. We recently tested two experimental vaccines; a subunit vaccine, which included two R. rickettsii recombinant proteins (RCA) and a whole cell inactivated antigen vaccine (WCA), to confer protection against virulent R. rickettsii infection challenge. WCA offered complete protection against RMSF, while RCA did not. This prior published work offers a strong scientific premise for the proposed detailed investigation. In particular, we aim to further characterize WCA in determining A) the duration of protection, B) the role of adjuvants in defining protection, C) the type of immune response observed, and D) the protection against tick transmitted homologous and heterologous challenges. We believe that this project, supported by strong scientific premise, addresses a significant public health problem. The goals are innovative as we will be the first group to investigate RMSF vaccine development using a physiologically relevant animal- tick-pathogen infection model. The central hypothesis of our application is that WCA protects against lethal RMSF caused by blood- and tick-borne infections, resulting from geographically distant pathogen strains, by stimulating immune protection for one year or longer. The specific aims of this application are: 1) Evaluate inactivation methods for preparing WCA and adjuvants in defining the vaccine protection. 2) Evaluate WCA protection against tick-transmitted challenges. 3) Evaluate WCA protection against R. rickettsii heterologous strain infection challenges. At the conclusion of this project, we expect that our efforts will translate to a fully developed vaccine, which is efficacious in an animal model known to naturally acquire R. rickettsii infections from an infected tick leading to life-threatening RMSF. We believe that achieving goals of this application will pave the way for extending vaccine studies to protect people from this lethal disease in the very near future.