University Of Missouri-Columbia

NIH Research Projects · FY 2025 · 2020-05

The ability to accurately predict protein interactions is critical for the mechanistic investigation of many biological processes and for rational therapeutic development. Over the past decades, methods ranging from physicochemical-based approaches to template-based and machine learning-based approaches have significantly advanced protein complex structure prediction. With the power of recent tools like AlphaFold2 and AlphaFold3, enormous progress has been achieved. Yet, there are several unmet, important challenges that remain unsolved beyond the existing computational methods, including AlphaFold. We will take advantage of the flexibility of the five-year R35 funding mechanism to address five different challenges under the umbrella of a common theme: developing novel computational methods to predict protein interactions and their complex structures by integrating principles of physics, bioinformatic approaches, and machine learning technologies. I will also continue to collaborate closely with experimental colleagues at our university, validate our computational methods and predictions, and explore potential therapeutic applications for the treatment of diabetic wounds. Goal #1: Predicting disordered protein-protein interactions. Goal #2: De novo design of peptides targeting the protein-protein interaction (PPI) interface. Goal #3: Development of novel peptide inducers of NRF2 that target the Keap1-NRF2 interface with potential applications for treating diabetic wounds. Goal #4: Development of a Graph Neural Network (GNN)-based deep learning algorithm for antibody-antigen interface True/False classification and multiple point mutation assessment. Goal #5: Development of GNN-based polarized protein-specific charges for protein-ligand docking and simulations. During the past four years since the funding of this R35 in May 2020, my lab has published 20 peer-reviewed papers supported by this grant on predicting protein interactions and lead discovery, including papers in high- impact journals such as PNAS and Nature Communications. I have additional five research manuscripts submitted to peer-reviewed journals (currently under review). We have also filed three pharmaceutical patent applications and obtained one software copyright, along with three webservers. Our software has been freely distributed to academic users from 22 countries across every continent. My team consistently ranks among the top in prestigious and highly competitive international competitions for blind protein-protein and protein-ligand structure predictions. All source codes developed from this project will be freely accessible to academic users and all webservers are freely accessible to everyone.

NIH Research Projects · FY 2025 · 2020-04

Project Summary The past five years have ushered in a transformative era in biomedical research. With the development of life- saving SARS-CoV-2 mRNA vaccines, FDA approval of the first RNA-targeted drug, and FDA approval of the first CRISPR-based gene editing therapy, RNA is revolutionizing key areas of medicine. Despite these remarkable advances, the full potential of these technologies remains untapped, partly due to bottlenecks in quantitative RNA modeling and design. While machine learning (ML) has profoundly influenced biomolecular modeling, it has yet to capture the complexities of RNA's heterogeneous conformational distributions, alternative folds, and kinetics — these are the hallmarks of RNA structure and function. Recent advancements in ML and the ever- growing data from RNA-based technologies have now positioned us to develop new biophysical tools and make significant breakthroughs in understanding, modeling, and designing RNAs. Leveraging the extensive structural and biophysical data, including over 500,000 thermodynamic and structural probing data points for more than 100,000 designed RNA sequences from experimental collaborators, we will develop hybrid approaches that integrate cutting-edge machine learning techniques with biophysical methods. We aim to develop tools targeting significant problems in RNA biology — from the prediction of RNA conformational distributions, RNA-small molecule interactions, and cotranscriptional folding to the learning and large-scale modeling of structure-based mRNA vaccine design. The new tools developed in this grant will provide a foundation for future designs of mRNA vaccines, antiviral drugs, and CRISPR genome editing.

NIH Research Projects · FY 2025 · 2020-03

Impact of repeated vaccination on the effectiveness of seasonal influenza vaccines Summary Influenza viruses cause pandemic and seasonal outbreaks that lead to the loss of thousands to millions of human lives. Vaccination is the best option for preventing influenza outbreaks and minimizing their effects on health. In the United States, annual influenza vaccination has been recommended since 2010 for persons 6 months of age and older. However, vaccine performance varies significantly between influenza seasons, and reduced vaccine effectiveness has been observed. Studies have reported that persons vaccinated during two consecutive influenza seasons had lower vaccine effectiveness during the second season than persons who had not been vaccinated during the prior season. These findings have caused profound confusion among the public regarding the potential benefit of annual influenza vaccination. Thus, there is a critical need to address the effect of repeated vaccination–associated pre-existing immunity on influenza vaccine performance. The objective of this project is to characterize the effects of repeated influenza vaccination on the specificity and magnitude of cross-reactive antibodies and on the effectiveness of seasonal influenza vaccines. Two specific aims are proposed: 1) determine the specificity, magnitude, and longitudinal patterns of humoral responses in humans with repeated seasonal vaccination, and 2) test the effect of repeated vaccination– associated pre-existing immunity on influenza vaccine performance in ferrets. By comparing antibodies in persons with and without repeated influenza vaccination, we expect to show whether and how pre-existing immunity achieved through repeated influenza vaccination affects the specificity and magnitude of the cross-reactivity for the resulting antibodies and, thus, vaccine effectiveness. From our studies in ferrets, we expect to show whether and how variations in repeated vaccination–associated pre- existing immunity affect influenza vaccine performance. This study will expand our understanding of molecular mechanisms that may influence how repeated vaccination affects influenza vaccine performance. Thus, this study will provide basic knowledge for evaluating the need for annual influenza vaccination and for optimizing influenza vaccine performance.

NIH Research Projects · FY 2025 · 2020-02

The University of Missouri-Columbia (MU) proposes to continue an IMSD T32 training program for doctoral students from groups that are underrepresented (UR) in biomedical research to become successful scientists pursuing careers in the biomedical workforce. The mission of our training program is to facilitate the transformation of our trainees into independent scientists who are active producers of new biomedical knowledge and are adept at solving the complex problems of disease and illnesses that adversely affect human health. Our training program accomplishes this mission by bringing together four PhD programs including Biological Sciences, Biochemistry, Biological Engineering, and Translational Biosciences. The latter program includes parallel emphasis areas (tracks) covering specialties including microbiology & virology, gene & stem cell therapy, immunology, physiology, nutrition & exercise science, cancer biology, and epidemiology & precision health. We propose to use science identity as our organizing principle, with its composition of community belonging, self-efficacy, and achieved deliverables, and its special note in the literature that UR trainees, more than non-UR trainees, may be particularly responsive to programmatic improvements in this arena. Objective #1: To increase participation of UR students in four doctoral programs from the current level of 20% to 30%. Objective #2: To strengthen the inclusive and supportive nature of our training community by providing training to improve the quality of the peer- and mentor-mentee relationships that are so critical to career success and satisfaction. Objective #3: To expedite fluency in the scientific method. We will institute a Critical Thinking Workshop to be followed by a new course, Biomedical Literature Colloquium, for primary literature critique, presentation, and writing. Objective #4: To promote professional communication skills and increase achieved deliverables in the form of external fellowship wins and peer-reviewed publications. Objective #5: To improve the PhD completion rate of IMSD trainees in the participating biomedical doctoral programs from the current level of 74% to 85%. Our active program evaluation structure will allow independent, professional assessment of trainees’ sense of science self- efficacy, science identity, perceived supports, and satisfaction with mentoring relationships, all of which are predictors of academic satisfaction and persistence outcomes among students from UR backgrounds. The IMSD T32 program will implement a wide-ranging set of programmatic activities to develop the technical, operational, and professional skills that will enable our UR trainees to flourish in their independent scientific careers.

NIH Research Projects · FY 2026 · 2019-08

Project Summary Parthenogenesis (i.e., reproduction without mating) has evolved from sexual reproduction in nearly all major eukaryotic groups. In parthenogenesis, chromosomally unreduced (e.g., diploid) gametes result from modified forms of meiosis. Understanding the genetic mechanisms underlying the modification of meiosis in parthenogenetic lineages is of significant public health interest because meiosis is central to sexual reproduction. Using parthenogenesis to understand the genetic regulation of meiosis is also a highly innovative approach, with its natural history perspective most likely yielding novel knowledge about meiosis. Using a combination of evolutionary and functional genomic approaches, this project examines the genetic bases of cyclical and obligate parthenogenesis in the freshwater microcrustacean Daphnia. Daphnia is well known for its cyclical parthenogenesis (CP) life cycle, i.e., propagating asexually under favorable environmental conditions and switching to sexual reproduction in response to stressful environment. Interestingly, some populations of the species D. pulex (backcrosses of two parental CP species D. pulex and D. pulicaria) reproduce by obligate parthenogenesis (OP) because they lost the capability to engage in sex. My lab has two long-term goals. First, considering that environmental conditions can trigger CP Daphnia to switch between parthenogenetic and sexual reproduction, environment-mediated gene expression changes most likely play the role of master regulator for reproductive mode. Our recent transcriptomic analyses revealed the many paralogous genes in the Daphnia genome show divergent expression associated with reproduction mode. For example, one paralog is upregulated in parthenogenesis, whereas the other paralog is upregulated in sexual reproduction. These paralogous contain multiple transmembrane receptors, neurotransmitter receptors, and transcription factors. We therefore hypothesize that these paralogs contain the master regulator of cyclical parthenogenesis. To identify the master regulator(s), we plan to use forward genetic screening to identify mutants that are not able to switch reproductive modes and identify the causal genes using functional analysis such as CRISPR gene editing. We will also directly perform gene knockout experiments to examine the functions of the divergently expressed paralogs in relation to reproduction in CP Daphnia. Second, concerning the origin of OP, we hypothesize that incompatible genetic elements between the two parental species CP D. pulex and CP D. pulicaria are responsible for the loss of sexual reproduction. To identify the causal incompatible elements, our lab will perform large-scale association mapping on newly discovered D. pulex crosses (between CP D. pulex isolate carrying D. pulicaria introgression) that produce both OP and CP F1s. The identified candidates will undergo functional genomics analysis (e.g., CRISPR gene knock-out), with the goal of re-creating OP animals by introducing OP-causal elements to CP Daphnia using gene editing. Lastly, we will examine whether the identified elements are present in natural OP isolates to understand whether multiple genetic mechanisms underlie the origin of OP.

NIH Research Projects · FY 2024 · 2019-07

Use of Clinical Samples to Identify Influenza Virus Antigenic Variants Summary Influenza A viruses (IAVs) cause pandemic and seasonal outbreaks that lead to the loss of thousands to millions of human lives. Vaccination is the best option for preventing influenza outbreaks and minimizing their effects. An understanding of the antigenic evolution of influenza viruses and the rapid selection of a well- matched influenza vaccine strain is the key to developing an effective vaccination program. However, antigenic characterization for influenza viruses presents two great challenges: 1) virus propagation, which is required in conventional serologic assays, can cause culture-adapted mutations and skew antigenic properties of viruses in clinical samples, and 2) reference sera used in conventional serologic assays are produced in influenza virus–seronegative ferrets and do not represent the immunologic profiles of human serum, which often has had prior exposures to influenza viruses through vaccination, natural infection, or both. An ideal platform for determining antigenic properties of influenza viruses and for selecting influenza vaccine strain should directly use clinical samples. The objectives of this project are 1) to develop and apply a novel high-throughput technology to directly characterize antigenic properties of influenza viruses by using human clinical samples without virus isolation and propagation and 2) to understand antigenic evolution of IAVs by using clinical samples directly. The antigenic characterization will include influenza virus–positive clinical samples from which virus can or cannot be cultivated. To understand influenza virus quasispecies in clinical samples and the effect of culture-adapted mutations on antigenic characterization, we will perform next-generation genomic sequencing on the clinical samples and corresponding isolates. We will then study the effects of the sequence diversity on antigenic variations of influenza viruses. We will also determine the effect that prior exposure to influenza virus(es) has on antigenic characterization during influenza vaccine strain selection. This project will help us provide fundamental technology for characterizing the antigenicity of influenza viruses in clinical samples without propagating virus. The resulting platform for antigenic characterization will overcome biases arising from virus propagation in conventional serologic assays. In addition, this is a high- throughput method and will significantly reduce the human labor needed for serologic characterization, decrease the time required for antigenic characterization, and increase the number of samples in antigenic characterization. Thus, this project will lead to significant technologic advances in influenza vaccine strain selection and facilitate influenza prevention and control. In addition, this project will provide knowledge about molecular mechanisms in antigenic variations associated with influenza virus quasispecies and genomic diversity and knowledge about prior human exposure to influenza viruses, which will help us optimize antigenic characterization in vaccine strain selection and understand antigenic evolution of influenza viruses.

NIH Research Projects · FY 2025 · 2019-06

PROJECT SUMMARY The Veterinary Medical Diagnostic Laboratory (VMDL) at the University of Missouri (MU) is a full- service for all animal species diagnostic laboratory and it is Missouri’s only laboratory accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD). The VMDL is a Tier I Laboratory of the Food and Drug Administration (FDA) Veterinary Laboratory Investigation and Response Network (Vet-LIRN). In addition, the VMDL is a Level I Lab of the National Animal Health Laboratory Network (NAHLN). The VMDL provides in-depth laboratory diagnostic support to MU Veterinary Teaching Hospital, non-MU veterinary practitioners, livestock and poultry industry, stakeholders in companion animal health, wildlife conservationists, and state and federal regulatory agencies. The goal of this application is to maintain the VMDL’s strong support to FDA Vet-LIRN mission in investigating potential adverse events affecting the nation’s food or animal feed supply by testing veterinary products, animal feeds or diagnostic samples. The specific aims are to 1). participation in FDA/Vet-LIRN sample analysis, 2). provide analytical data to support potential regulatory use, and 3). participate in additional projects, such as method development and validation as determined by the VPO. The infrastructure funding will enable the VMDL to better support FDA CVM’s capacity and capability to conduct case investigations.

NIH Research Projects · FY 2026 · 2018-09

PROJECT SUMMARY Preeclampsia is a major source of maternal and fetal morbidity and mortality in pregnancy. Multiple studies have documented abnormalities in placental trophoblasts obtained from preeclamptic pregnancies at term, in development and function of both syncytiotrophoblast (STB) and extravillous trophoblast (EVTB). These cell types arise early in gestation, and it is unclear whether the abnormalities contribute to the pathophysiology of preeclampsia or are only a result of the preeclamptic environment. The proposed project will use three innovative models to address this gap in knowledge: (1) Induced pluripotent stem cells (iPSC), derived from control and preeclamptic pregnancies and differentiated to a mixed population of peri-implantation stage trophoblast by exposure to BMP4, and signaling inhibitors; (2) Induced trophoblast stem cells (TSC), obtained by conversion of the control and EOPE iPSC lines, and differentiated to the first trimester, villous stage of placental development, along either the STB or EVTB lineage; (3) 3D trophoblast organoids, derived from the TSC lines, and differentiated to predominantly STB or EVT. There are three specific aims: Aim 1 will test whether STB differentiation and, as a consequence, function, is defective in EOPE in the various models of first trimester placenta. Aim 1A will test the hypothesis that defects in STB assembly and function will be discernible in EOPE in the models of peri-implantation and villous STB developed in this laboratory, particularly under conditions of oxidative stress. In Aim 1B, single nucleus RNAseq will be used to distinguish whether TB types, especially the two previously identified clusters of 8TB, are differentially affected. Aim 2 will characterize EVTB differentiation and invasive properties in normal pregnancy and EOPE by using the TSC models. Aim 2A will determine whether defective TB invasion in EOPE is characteristic of first trimester-like EVTB, as it is in peri-implantation TB. Aim 28 will utilize organoid cultures to assess EVTB differentiation in 3-dimensional space, in homology to anchoring villi, and the role of cell-matrix interactions in acquisition of specific EVTB subtypes. Aim 3 will determine whether mitochondrial defects affect EVT and STB in EOPE Aim 3A will determine whether diminished ATP production reduces invasion in EOPE under stressful conditions. Aim 3B will test whether EOPE TBs are more susceptible to stress-induced production of reactive oxygen species. Accomplishment of these aims will uncover the mechanisms behind impaired STB and EVTB differentiation and function in patient-derived culture models of first trimester placental development.

NIH Research Projects · FY 2025 · 2018-06

Abstract Youth current e-cigarette use rates have dramatically increased in recent years, reaching 19.8% in 2020. State and municipal governments have passed flavor bans, e-cigarette taxes, and Tobacco 21 laws in part to respond to these high rates of youth e-cigarette use. The long-term goal of our research agenda is to provide information that will allow for optimal regulation of e-cigarettes by identifying intended and unintended effects of e-cigarette policies. Specifically, the objective of our current renewal application is to use quasi-experimental methods (e.g., difference-in-differences) to evaluate contemporary tobacco policies of flavor bans, Tobacco 21 laws, and new e-cigarette tax schema. We will use high quality, reproducible data from the Truth Longitudinal Cohort Study, Population Assessment of Tobacco and Health, Monitoring the Future, and Youth Risk Behavior Surveillance System to estimate: (i) the effect of flavor bans with varying levels of strength on youth e-cigarette use, combustible tobacco product use, and substitution towards the remaining legally available flavored tobacco products; (ii) the effect of Tobacco 21 laws on youth e-cigarette use, combustible tobacco product use (e.g., cigarettes, cigars, hookahs), and source of e-cigarettes; and (iii) the effect of e-cigarette taxes, including new e-cigarette tax schema of sales taxes and two-tier taxes, on youth e-cigarette use and combustible tobacco product use. Our project is significant by carefully examining high rates of youth e-cigarette and alternative combustible tobacco products in the United States; identifying policies to reduce tobacco-related disease and death (the leading cause of preventable death in the United States); by contributing externally- valid estimates of the effect of flavor bans from as many as 330 state and municipality flavor restrictions currently on a wide variety of both flavored and unflavored tobacco product outcomes; by carefully considering the strength of flavor bans including products covered, flavors covered, and retailer exemptions; by considering questions on tax saliency for new types of e-cigarette taxes; and by being among the first to study the effect of Tobacco 21 laws on youth e-cigarette use and where youth obtain e-cigarettes. Our project is innovative by using cutting-edge quasi-experimental methods designed to mimic a randomized control trial including newer methods designed for the presence of heterogenous treatment effects; being the first to use Truth Longitudinal Cohort study data and Population Assessment of Tobacco and Health data for quasi-experimental research; by providing a database of standardized e-cigarette taxes; by providing a workshop on heterogenous treatment effect methods; and by collaborating across economics departments, a leading tobacco control non- governmental organization (Truth Initiative), and schools of policy, public health, and medicine. Truth Initiative will lead our dissemination efforts using their extensive outreach and advocacy infrastructure.

NIH Research Projects · FY 2025 · 2018-05

Project Abstract The breadth and depth of deep learning (DL) in solving fundamental biological problems have been demonstrated. DL-based approaches, such as AlphaFold2 for 3D protein structure prediction, have become widely accepted by the biology community. The Xu lab has been at the forefront of developing novel DL algorithms, software, and information systems for diverse biological and medical problems. During the current project period, the Xu lab has made excellent progress in addressing some of the urgent challenges and needs for developing DL methods in biological sequence analyses and predictions, as well as other bioinformatics problems. This R35 project has produced 31 papers covering research topics ranging from protein sequence- based predictions to drug design, molecular dynamics simulation, and single-cell data analysis. In addition, it also provided more than ten open-source tools and three major web-based resources to the community. The rapid development of new DL techniques and Xu lab’s accumulating expertise in this field bring new opportunities in shaping DL to molecular biology. The current widely used supervised DL methods in biomedical research often do not have sufficient data with clean and accurate labels for training and may not have good generalizability. The emerging self-supervised learning (SSL) approaches that aim to learn informative representations by exposing relationships between different data perspectives without human annotations are becoming a new trend. Different data perspectives are broadly called multiview. The multi-view SSL techniques allow us to generate joint or coordinated representations for single modal and multimodal data with stronger generalizability, better robustness, and less bias. Though SSL has demonstrated great successes in other fields, it has only been minimally explored in biology. This renewal project will develop a multi-view SSL framework that can handle both single-view and multi- view data and is capable of single and multiple tasks. It will tackle key challenges and bottlenecks in applying SSL for biological studies, such as selecting effective views and data augmentations, fusing multimodal data or data from heterogeneous sources, and integrating biological constraints into SSL models. We will focus on designing a biology-informed system, enhancing generalizability and robustness, and making the results biologically interpretable and confidence assessable. The Xu lab will apply and refine the framework to multiple mainstream biology applications, including anti-CRISPR protein prediction, by exploring various data augmentation methods for protein sequences, ion and small ligand binding prediction using complementary views of protein sequences and structures, and single-cell data analyses across different conditions. The framework will also be tested for broad applications in sequence-based studies and beyond, such as alignment- free methods for constructing phylogenetic trees and detecting novel protein families, as well as conducting cross-species single-cell data analysis.

NIH Research Projects · FY 2024 · 2017-08

Abstract: Current data indicate that that purinergic signaling can potentially affect every cell in the human body. Indeed, one theory is that extracellular ATP (eATP) is the oldest, extracellular signal involved in cell-cell communication. The mechanisms of purinergic signaling are well-established in animals and, indeed, support a multibillion dollar pharmaceutical industry. In contrast, relatively little is known about purinergic signaling in plants. Plants do not possess canonical P2X an P2Y purinoreceptors. Indeed, our lab previously identified a new class of purinoreceptors in plants, exemplified by plasma membrane lectin-receptor-like-kinases, P2K1 and P2K2. Our research indicates that purinergic signaling in plants is as ubiquitous and impactful as that found in mammals and, indeed, many of the downstream effects are similar. The differences seen, comparing plants and animals, are due largely to the unique biochemistry of the P2K receptors. These are unique receptors in that they possess both kinase and nucleotide cyclase activity. Hence, the primary aim of this proposal is to explore further the function of these receptors and, specifically, the relative contribution of these two activities to cellular response to eATP. Specific aim 1 will address the hypothesis that ATP released from plant cells activates both P2K1 nucleotide cyclase and kinase activity, which subsequently mediate distinct downstream, cellular responses. Study 1 will utilize mutant studies of P2K1 to define the relative contribution of these two activities to downstream signaling responses. Study 2 will explore the relative importance of P2K1 cyclase and kinase activity relative to the activation of cyclic nucleotide gated calcium channels (CNGC). Study 3 will examine whether activation of the cytoplasmic kinase, BIK1, via P2K1 phosphorylation leads to CNGC Ca2+ channel activation. Specific aim 2 will address the hypothesis that, as is the case in animals, plants likely have multiple purinoreceptors that may act in specific tissues, during specific stages of development or in response to specific stresses. Preliminary data argue that additional purinoreceptors exist in plants. Hence, Study 4 will seek to identify additional plant purinoreceptors using a variety of approaches. Most notable is our finding that mutations that suppress the phenotypes of p2k1 mutants do so in the apparent absence of any known purinoreceptor, clearly indicating that other mechanisms must exist. Preliminary data suggest that purinoreceptors mediate the negative effects of stress on plant growth. Study 5 will explore how wounding stress is coupled to a reduction in growth by exploring the role of an atypical basic-helix-loop-helix protein, previously implicated in growth regulation, that is a direct target of P2K1 phosphorylation. The net result of our work is to provide the comparative data to add to our overall understanding of purinergic signaling in higher eukaryotes, illustrating differences and similarities, and ultimately laying the basis for opportunities to manipulate these pathways for therapeutic applications.

NIH Research Projects · FY 2025 · 2017-08

Project Summary / Abstract Adeno-associated virus (AAV) is a leading delivery vector for gene therapies of a wide range of genetic disease and predilections. The FDA has recently approved AAV-mediated treatments for spinal muscular atrophy (SMA) and RPE-associated retinal dystrophy, while 150 clinical trials are ongoing. However, inefficient transduction requires high doses with risk of immunotoxicity that has been evident in clinical trials. A genome-wide screen for host proteins, needed for AAV cell transduction, changed the understanding of AAV entry. Hitherto-uncharacterized AAVR was identified as a key receptor, while the downstream role of GPR108 is most likely in endosomal escape. AAVR cryo-EM structure will be put into biological context. Studies of GPR108 will start by pinpointing the entry step and cell location where AAV and GPR108 associate, and identification of the domains with which AAV interacts. The approach to structure will be holistic. Overall configuration will come from cryo-electron tomography (ET) of AAV complexed with native-like GPR108 nanodiscs. This will be integrated with high resolution cryo-electron microscopy (EM) of AAV complexed with expressed extracellular domains of GPR108. Triggers will be investigated of an AAV conformational transition that releases a virally-sequestered phospholipase A2 for endosomal escape, as AAV traffics to the nucleus. Antibody neutralization of AAV is widely considered mediated by interference with glycan-binding or of a post-entry step. This will be reevaluated after finding overlap in the binding of AAVR and several neutralizing monoclonal antibodies. Prevalent in vivo modes of binding will first be established through cryo-EM using polyclonal antibodies from pooled human serum. Mutations, directed towards escape, will be used to test whether receptor-binding and entry are, in fact, primary mechanisms of neutralization, and whether escape to rapid neutralization by pre-existing antibodies can be designed without harm to receptor-mediated cell entry. Computer methods, optimizing atomic structure vs. cryo-EM maps, prototyped during AAV studies, will be developed and disseminated. Modules will implement the RSRef algorithm, unique in matching to map values, a sum of 3D atomic profiles of refinable experimental resolution. This will be combined with open source force fields, opening an emerging application of atomic resolution cryo-EM in determining hydrogen bonding networks and protonation states. For resolutions below 3 Å, restraints eliminating unnecessary freedom will be introduced to limit the over-fitting that is an under-appreciated problem in EM refinement. The project’s main goal is a molecular understanding of AAV’s host interactions, providing structural and mechanistic foundations for engineering needed improvements in vector efficiency and specificity in development of safe gene therapies. Broader collateral impact will come from a model system for viral entry that will be put to rigorous practical test, and computer methods for improving the precision of EM structures.

NIH Research Projects · FY 2026 · 2017-03

Project Summary/Abstract Transient receptor potential vanilloid, member 4 (TRPV4) is a cation channel highly expressed in cardiomyocytes with aging, and contributes to enhanced cardiomyocyte calcium cycling and hypercontractility following TRPV4 gating stimuli including osmotic stress and mechanical stretch. Excessive TRPV4 activation leads to cardiac damage and ventricular arrhythmia. Angiotensin II (AngII) is a peptide hormone critically important to cardiovascular physiology and pathology due to its regulatory effects on blood volume and pressure. In many cell types, AngII increases TRPV4 activity although the contribution of this signaling axis to cardiomyocyte calcium homeostasis is currently unknown. This renewal proposal tests the central hypothesis that TRPV4 contributes to AngII-dependent cardiomyocyte calcium signaling and ventricular arrhythmia. To test this hypothesis, we will use both a pharmacologic (TRPV4 inhibition) and genetic approach (cardiomyocyte specific TRPV4 deletion or overexpression) to examine the functional role of TRPV4 in isolated cardiomyocytes, in isolated perfused hearts, and in mice in vivo. Specific Aim 1 uses isolated cardiomyocytes and isolated perfused hearts to test the hypothesis that AngII promotes TRPV4 trafficking, increases TRPV4 activity, and enhances cardiomyocyte calcium transients during excitation-contraction coupling. Specific Aim 2 tests the hypothesis that cardiomyocyte TRPV4 contributes to AngII-induced cardiac remodeling, and examines the role of TRPV4 in hypertrophic and fibrotic remodeling following AngII excess (osmotic mini-pumps) and during biological aging. Specific Aim 3 tests the hypothesis that TRPV4 contributes to pro-arrhythmic cardiomyocyte calcium signals and ventricular arrhythmia following both acute and chronic AngII excess. The overall goal of this project is to establish TRPV4 as a therapeutic target to prevent arrhythmia with aging.

NIH Research Projects · FY 2026 · 2015-09

Project Description Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle-wasting disease caused by dystrophin deficiency. Adeno-associated virus (AAV)-mediated systemic microdystrophin (μDys) gene therapy aims to treat DMD with a bodywide expression of abbreviated dystrophin. With the support from NIH, we have made many important contributions to the development of AAV μDys gene therapy. We pioneered AAV μDys therapy for DMD cardiomyopathy; we were among the first to show that AAV μDys therapy can improve muscle force in the mouse model; we were the first to demonstrate the histological and physiological benefits of AAV μDys therapy in a large dystrophic mammal (the canine DMD model) by intramuscular injection; and we were the first to achieve successful bodywide AAV μDys therapy in the canine DMD model. In addition, we discovered several domains important for μDys function, including the dystrophin nNOS-binding domain and membrane-binding domains. These studies and the outstanding contributions from many other laboratories have led to systemic AAV μDys trials in DMD patients by several biopharma companies and the recent approval of an AAV μDys drug by the FDA. Despite encouraging progress, several important issues surfaced in clinical trials. These include toxicity associated with high-dose systemic AAV delivery, low efficacy and immunogenicity of current μDys constructs, and uncertainty of the longevity of the therapy. The field is also hindered by the lack of a good method for predicting AAV performance in human muscle. The canine model is the most established large animal model. It faithfully recapitulates human disease. We are a world-leading laboratory developing DMD gene therapy in the canine model. We recently developed a xenograft model for testing systemic AAV delivery in human muscle. In this renewal, we will leverage our expertise in AAV μDys therapy, the canine DMD model, and the human muscle xenograft model to (1) address whether the toxicity associated with the high-dose AAV administration can be attenuated with the newly developed more potent myotropic AAV capsids; (2) determine whether myotropic AAV μDys therapy can result in long-term therapeutic benefits in the canine DMD model; (3) identify the best-performing AAV capsids for systemic gene therapy in human muscle, and (4) develop more potent, deimmunized next-generation μDys constructs. Our studies will greatly advance AAV μDys gene therapy for DMD and reduce the disease burden on patients, families, and society.

NIH Research Projects · FY 2025 · 2015-07

This application proposes to continue, refine, and disseminate a 9-week summer research program in alcohol and addiction research. The program, the “MU Alcohol Research Training Summer School” (MU-ARTSS) at the University of Missouri, is targeted at undergraduate students with the goal of preparing them for graduate training in health-related scientific disciplines focusing on alcohol and addiction research. The program recruits seven students annually drawn from a national pool of applicants. MU-ARTSS begins with a one-week intensive set of didactic lectures on alcohol research. Topics include: introduction to addiction research, epidemiology, genetics, neuropharmacology, neurophysiology, individual differences, assessment and treatment, responsible conduct of research, and human subjects issues. In addition, trainees attend demonstrations of addiction-relevant research protocols, including mobile and ambulatory assessment, structural and functional neuroimaging, brief interventions, and laboratory administration of alcohol to human subjects. Following this didactics week, students complete an 8-week internship in the lab of a mentor conducting alcohol research. During the internship period, MU-ARTSS trainees also attend a weekly brownbag series covering specific research topics and professional development issues and a weekly “journal club” to help extend and integrate the broader learning that occurs during the initial didactic portion into the rest of the summer experience. Additionally, a weekly “movie series” is hosted by program mentors showcasing notable films about alcohol and addiction to allow for less formal discussion and exploration of clinical phenomena related to alcohol and addiction and the role and portrayal of alcohol in society. MU-ARTSS programming is supplemented by additional experiences offered by the MU-Summer Undergraduate Research Program (MUSURP). Partnering with MU-SURP affords our trainees an opportunity to interact with research faculty and students across multiple scientific disciplines and in multiple venues including professional development mini-conferences, further instruction in the responsible conduct of research, a seminar series, and a formal end-of-the-program poster session. MU-ARTSS draws on the large number of active alcohol research programs in MU’s Department of Psychological Sciences. The focus of MU-ARTSS on undergraduate education also provides an important complement to the alcohol training emphasis at the graduate and postdoctoral levels (currently supported, in part, by a T32 to Co-Director McCarthy). To date, 41 of 53 MU-ARTSS interns have completed their undergraduate degrees, and of these, 31 have enrolled or completed graduate or MD programs. The current proposal aims to continue refining MU-ARTSS recruitment and curriculum activities, and importantly, increase dissemination of materials and outcome data produced by the program.

NIH Research Projects · FY 2026 · 2013-05

Abstract α-Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The chaperone-like activity of α-crystallin, which was discovered nearly three decades ago, is implicated as a key component in the maintenance of lens transparency by suppression of crystallin aggregation. It was found that the deletion of 21-28 and 54-61 regions of αB-crystallin leads to increased chaperone-like activity (activation, gain of function). Understanding the molecular organization and properties of crystallin subunits in activated chaperones would help answer questions on how α-crystallin chaperone-like activity might be harnessed and manipulated for the development of protein-based therapeutics. It is hypothesized that the increased αB- crystallin chaperone-like activity in deletion mutants stems from new type of oligomers where subunit–subunit interactions lead to the exposure of “cryptic” chaperone sites in the native oligomers. Studies show a recombinant αB-crystallin expressed after deleting either 54-61 or 21-28 and 54-61 sequences (resulting in a protein designated as αBΔ54-61 and αBΔ21-28,Δ54-61) was found to form smaller oligomers than the wild- type protein but to show up to ~25-fold increase in chaperone-like activity. The experiments proposed in this proposal will uncover the molecular changes that drive the increased chaperone-like activity in αBΔ21-28,Δ54- 61 and αBΔ54-61. The aims of the application are 1) Uncover the molecular changes in the activated αB- crystallins, (αBΔ54-61 and αBΔ21-28,Δ54-61), 2) determine the biological implications of enhanced chaperone-like activity of engineered proteins in the cell culture and whole lens culture system. Novel crosslinker(s) will be used to gain fresh insights into the “cryptic” chaperone sites getting exposed in the activated crystallin. The studies will also make use of site-directed mutagenesis and mass spectrometric analysis to uncover the molecular changes at subunit interaction level in activated oligomers. To see whether the activated αB-crystallin can be exploited to protect cells from oxidative injury, the effects of stress-inducing agents such as H2O2 and sodium iodate will be investigated in HEK293 and ARPE-19 cells in presence of activated crystallins. Further, the ability of activated chaperones to suppress aggregation and toxicity of fibril- forming β-amyloid will be investigated both in vitro and ex-vivo. The long-term goals of the studies are to understand the structure–function relationship of activated αB-crystallins and develop crystallin proteins that have therapeutic value in protein conformational diseases and oxidative stress conditions.

NIH Research Projects · FY 2025 · 2012-07

ABSTRACT According to the American Cancer Society, more than 60,000 people will develop head and neck cancer this year and those patients must receive radiation therapy to survive. This treatment regularly destroys the salivary glands (SG), leading to a loss of secretory function which is typically permanent. Current treatments remain largely ineffective, with therapeutic interventions being limited to use of saliva substitutes with modest effectiveness and medications that provide only temporary relief. In light of the high degree of need and the limitations of current therapies, development of alternative treatments to restore SG functioning is essential. In response to the challenges noted above, we propose introduction of FGF7 and FGF10, both of which activate FGF2b signaling to promote SG epithelial morphogenesis and differentiation (Aims 1 and 2, in vitro and in vivo, respectively). Having fortified our scaffold to enhance SG morphogenesis and differentiation, we will nonetheless still be faced with absent or poorly developed vasculature and nerve systems, as indicated by repeated studies demonstrating loss of vascularization and innervation in irradiated SG. In response to these challenges, we will draw on our previous findings indicating VEGF and FGF9 to aid vascular formation and neurotrophic factors (e.g., NGF) to promote innervation (Aim 3). We hypothesize that a modified FH scaffold containing immobilized L1 peptides (L1p) and GF (L1p-GF-FH) will promote formation of functional tissue in irradiated SG. Aim 1: will demonstrate sustained secretory function using a fortified scaffold in vitro. We will determine whether incorporation of FGF7 and FGF10 into the L1p-FH (termed Ep-FH) scaffold allows secretory function to remain intact for an extended duration in irradiated SG. Aim 2: will demonstrate sustained secretory function using a fortified scaffold in vivo. We will determine whether incorporation of FGF7 and FGF10 into the L1p-FH (termed Ep-FH) scaffold allows secretory function to remain intact for an extended duration in an irradiated SMG mouse model. Aim 3: will restore full functionality to irradiated SG in vivo. We will combine our Ep-FH scaffold with polymeric microparticles to release pro-angiogenic and pro-innervation GF in a temporal sequence, mimicking the in vivo physiology, to enhance functional recovery of SMG following radiation treatment. In summary, our proposed studies will extend our findings to date using L1p to restore SG function, thereby allowing for both greater sustainability and a deeper degree of functionality.

NIH Research Projects · FY 2025 · 2003-09

Project Summary The overall goal of this application is to continue and expand operation of the National Swine Resource and Research Center (NSRRC). The NSRRC is halfway through its 19th year of operation, and it has successfully developed the infrastructure needed to assist swine-based research across multiple disciplines. The NSRRC has developed new models of human disease by genetic engineering, it has recruited models created by others, and it has distributed expertise, cells, tissues, organs, and pigs to investigators throughout the United States and Europe. Since genetic engineering of pigs requires specialized facilities and expertise, the NSRRC provides invaluable services to the research community by creating new genetically engineered swine models. The NSRRC also provides training and education by interacting with individuals, publishing manuscripts and sponsoring workshops and meetings that promote the use of pigs for studying human diseases. The NSRRC has a state-of-the-art building with high biosecurity, to house animals free of specific pathogens. The Specific Aims for the upcoming grant period are as follows: 1. To operate the National Swine Research and Resource Center. Functions of the NSRRC will continue to include: a) importation of existing swine models of human health and disease, b) rederivation of pigs to eliminate specific pathogens, and health monitoring to assure maintenance of a specific pathogen-free status, c) cryopreservation and storage of gametes, embryos and somatic cells to prevent future loss of valuable models, d) distribution of expertise, reagents, cells, tissues, organs and pathogen-free pigs, and e) creation of new genetically- engineered pigs needed by the biomedical research community. Rigorous quality control will ensure that only high-quality genetics are distributed. 2. To perform innovative research that will benefit the NSRRC and the biomedical research community. Applied research projects are aimed at improving: a) the cryopreservation of pig reproductive cells and tissues, and b) the development of improved methods for the production of genetically-engineered pigs. The NSRRC will also serve as a site for training and educational activities related to research employing swine models.

NIH Research Projects · FY 2026 · 2002-08

PROJECT SUMMARY / ABSTRACT The University of Missouri-Columbia (MU) proposes to identify, provide financial support, and train a total of 35 students from groups dramatically underrepresented in the biomedical sciences in order to prepare them to successfully enter and complete a PhD program in a biomedical or biomedical-related discipline at MU or any other highly competitive research-intensive institution. This proposal presents a coordinated plan to strengthen the research, academic, and personal interaction skills of promising and talented baccalaureate graduates by immersing each Scholar in an independent research project with a faculty mentor and providing a coordinated academic and personal support system. Specific MU PREP components include design and completion of an independent research project; strong research mentoring by committed faculty, aided by prior training of faculty mentors; training in responsible conduct of research and scientific expression; training in critical thinking and analysis; peer and group mentoring; and presentation of their research results at national/international meetings in the discipline. PREP Scholars should thus begin their PhD programs with advanced research skills, with a faculty and peer mentoring system in place, with connections to a broader discipline beyond MU, and with lasting friendships with other graduate students in the life sciences. The MU PREP program will be carefully coordinated with MU's other undergraduate and PhD programs for underrepresented students thereby advancing MU's comprehensive effort to increase the number of scholars from underrepresented populations in the biomedical sciences. Successful parts of this program will be institutionalized and disseminated as a model for other institutions.

NIH Research Projects · FY 2025 · 2002-07

This competing continuation application seeks an additional five years of support for a pre- and post-doctoral training program in the psychology of alcohol and addiction. The training program is housed in the Department of Psychological Sciences at the University of Missouri-Columbia (MU) and has been led by Kenneth J. Sher since its inception in 2002. The program takes advantage of a concentration of alcohol and addiction scientists at MU, as well as affiliated addiction scientists at the Washington University School of Medicine (WUSM) and MU’s sister campus, the University of Missouri – St. Louis (UMSL). The program has had remarkable success in producing addiction scientists who have gone on to noteworthy careers at leading institutions. The program will be co-directed by Drs. McCarthy and Sher for the proposed award period. The 28 training faculty includes both Preceptors and Secondary Mentors, with Preceptors serving as primary mentor and coordinator of a trainee’s experiences. Trainees are mentored to develop the skills and competencies needed to become independent investigators, direct their own research programs, and serve as effective members of multidisciplinary research teams. Trainees are drawn from Psychological Sciences, but receive training in the different research areas represented among the training faculty, including molecular and behavior genetics, behavioral pharmacology, nosology, cognitive neuroscience, ecological assessment, comorbidity, psychophysiology, community-based research, longitudinal research, clinical interventions, decision making, and quantitative methods. Training that integrates biological, psychological, and social factors is critical for advancing alcohol and addiction science, as the next generation of researchers must adapt to rapidly changing science and function effectively in multidisciplinary research teams. For the proposed period of award, we request to maintain our current level of 6 pre-doctoral and 3 post-doctoral trainees for all 5 years of support. Pre-doctoral trainees will typically have a two-year training period, while post-doctoral trainees will typically have a three-year training period. Pre-doctoral trainees are guaranteed a total of five years of support from the Department, and will be supported by other mechanisms (e.g., fellowships, research assistantships, F31 awards) in the remaining years of training. Post-doctoral trainees typically will move into faculty or research scientist positions at the conclusion of their period of support, usually at other institutions. Program specific training components include: 1) a formal course in alcohol and addiction studies, 2) a weekly program workshop, 3) required non-thesis/dissertation research, 4) a grant writing course and writing workshop, 5) attendance at regularly scheduled colloquia, 6) attendance at the annual RSA meeting, and 7) some exposure to alcohol-related clinical experiences (for some pre-doctoral trainees). Post-doctoral trainees typically have additional experiences tailored to fit their training needs. The training program is evaluated on an ongoing basis through both informal and formal means.

NIH Research Projects · FY 2026 · 2002-05

PROJECT SUMMARY Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical or covalent association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu and are instead channeled between enzyme active sites. Despite the widespread recognition that protein-protein interactions are ubiquitous, the molecular mechanisms of substrate channeling remain obscure, and the impact of channeling at the cellular and organismal levels is largely unknown. We seek to narrow these knowledge gaps by exploring substrate channeling within and between the enzymes of proline catabolism. The proposed experiments will explore long-distance, allosteric communication between the active sites of the bifunctional enzyme PutA using kinetic crystallography, assess the contributions of substrate channeling to bacterial fitness and pathogenesis, and determine the first structure of a novel bifunctional enzyme that moonlights as a transcriptional repressor using cryo-EM.

NIH Research Projects · FY 2026 · 2001-05

Abstract (Overall) The mission of the Rat Resource and Research Center (RRRC) is to provide biomedical investigators with the rat models, embryonic stem cells, related reagents, protocols, information, support and services that will facilitate their research. The RRRC serves as a unique repository by importing, storing and distributing a large number of rat strains/stocks. It assures that valuable models are preserved and made available to interested investigators, allows researchers to satisfy NIH requirements for resource sharing, relieves individual investigators from the burden of animal distribution and ensures that models are maintained with rigorous genetic quality control and health monitoring to promote experimental rigor and reproducibility. Recent advances in genome editing technologies are facilitating the ability to perform sophisticated genetic modifications in rats in order to study both normal biological processes and disease states. As a result, the number of rat models and the number of investigators using rat models for biomedical research is increasing and the RRRC, as one of the only centralized repositories in the world will continue to serve a critical role for archiving and distributing these new models. To improve repository functions, applied research to optimize methods for sperm cryopreservation and in vitro fertilization in the rat will be undertaken and ongoing efforts to refine, generate and characterize new genetically modified rat models needed by the research community will be performed. To expand the broad utility of the RRRC, we will continue to serve as consultants to the community and we will increase our fee-for-services in the areas of model generation, model characterization, colony management, cryopreservation and rederivation. By providing accessible expertise to investigators working with rats and by expanding our available materials and services, the RRRC will continue to serve as a much-needed “one stop” resource for investigators using rats in biomedical research.

NIH Research Projects · FY 2026 · 2000-05

PROJECT SUMMARY - OVERALL The Mutant Mouse Resource and Research Center (MMRRC) is the nation’s largest public mutant mouse archive and distribution repository organization. The primary goal is to facilitate research by identifying, acquiring, evaluating, characterizing, cryopreserving, and distributing mutant mouse strains to qualified biomedical investigators. The MMRRC was established by the NIH to ensure the preservation, dissemination, and development of valuable mutant mouse lines and data generated by research scientists. The MMRRC is a consortium of four Centers, each hosting an archive and distribution repository, and an Informatics Coordination and Service Center (ICSC) within a trans-national network regionally distributed across the United States. The Centers collectively serve the needs of the nation’s biomedical research community, ensuring access to and optimizing utilization of unique transgenic, knockout, and other genetically modified mutant mice and related biomaterials, services, and new technologies. The Centers collectively import, verify, maintain, and distribute mice, gene-targeted embryonic stem (ES) cells, and germplasm of genetically unique, scientifically valuable mice. Centers also provide services and procedures to assist investigators using genetically altered mice for research. Finally, Centers conduct resource-related research, often collaboratively, to further refine and develop mutant mouse lines to improve the reproducibility and translatability of mouse models for biomedical research. By depositing their mutant mice in repositories at the Centers, NIH-funded investigators fulfill their obligation under the NIH Sharing Policy. In return, each of the Centers strives to preserve the unique genetics, protect the germplasm, assess the genetic quality, and provide these models for the benefit of research scientists and investigators across the nation and the globe. In doing so, the MMRRC preserves the investment NIH made in these models and ensures that valuable mutant mouse lines are available across the biomedical research community, thereby accelerating the pace of rigorous reproducible research and discovery using genetically unique mice.

NIH Research Projects · FY 2025 · 1977-07

PROJECT SUMMARY Veterinarians, with their broad knowledge of animal biology are uniquely suited for advancing the field of comparative medicine. The University of Missouri Comparative Medicine Program is an established and preeminent training program that provides comprehensive research training for veterinarians with the goal of producing independent scientists conducting research in comparative medicine. The training provides a substantive foundation for a competitive research career through 1) an intense research experience to provide competence in state-of-the-art experimental methodology, 2) supporting course work and seminars that give a broad exposure to biomedical sciences, comparative medicine, experimental design, statistics, and biomedical ethics, 3) instruction in fundamental concepts of funding procurement and development of grant-writing skills, 4) instruction in all aspects of lab and project management including development of transferable skills, 5) instruction in scientific rigor and transparency in experimental design and study reproducibility with an emphasis on animal modelling, and 6) increasingly independent experience in every stage of the scientific research process. The strengths of this program include an exceptional mentor pool of over 40 well-funded faculty offering research opportunities in a broad range of areas related to comparative medicine, including but not limited to, infectious disease, genetics of disease, cryobiology and assisted reproduction, cardiovascular, renal and neurological function, cancer, and biomedical engineering. Furthermore, the presence of a National Mutant Mouse Resource Center, the only National Rat and Swine Resource Centers in the U.S., Metagenomics and Metabolomics Centers and one of 12 U.S. BL3-biocontainment facilities, provides a unique and unparalleled training environment for research and characterization of genetically engineered animals. Funds are requested to support four trainees for up to three years of research training under the mentorship of an established funded researcher. Trainees will design and perform a research project, prepare an extramural grant proposal, present research results at national meetings and publish their findings in high quality peer-reviewed journals. Training will culminate with preparation and defense of a dissertation (PhD). On completion of the training program, trainees will have acquired the skills needed to become successful independent investigators whose unique training will position them to become leaders in the Comparative Medicine community.