University Of Missouri-Columbia

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Cancer is set to bypass cardiovascular disease as the number one cause of death in United States and it is a leading cause of death worldwide. Non-Small Cell Lung Cancer (NSCLC) is a major contributing factor to this statistic. Recent advancements in chemotherapeutic delivery and the development small molecule inhibitors, such as tyrosine kinase inhibitors, have been indispensable in decreasing disease prevalence and burden. Additionally, the recent FDA approvals of immunomodulating therapies, such as immune checkpoint inhibitors (ICIs), chimeric antigen receptor (CAR) T cells, and bispecific antibodies, emphasizes the importance that immune system evasion plays in disease progression and relapse. Unfortunately, administration of these targeted and immunomodulating therapies is often met with tumor acquired resistance (e.g., secondary mutations; T cell exhaustion) and incites non-specific or on-target/off tumor side effects (e.g., immune related adverse events). This suggests a need for alternative or adjuvant NSCLC therapies that are not only potent and efficacious but exercise a wide therapeutic index. We propose to exploit aptamer technology as one potential way to address this need. Aptamers are single strand oligonucleotides that bind to their targets with high specificity and affinity and their relative lack of immunogenicity as a foreign substance, compared to antibodies, make them ideal reagents to modulate the immune system. Furthermore, their ease of manipulation makes molecular engineering to design and optimize such reagents relatively straightforward. Our goal is to develop novel immunomodulating bispecific aptamers (bsApts) that dually bind to immune cell CD3ε and NSCLC tumor associated antigens (TAAs) to induce formation of effective immune synapses. We propose to use molecular engineering techniques to rationally design bsApts and systematically evaluate specific bsApt properties, such as (i) valency, (ii) affinity, and (iii) linker length/type in their ability to induce artificial immune cell activation in vitro and anti-tumor responses in vivo. We also propose to take a transcriptomics approach to better understand how designs/targets affect tumor heterogeneity, tumor infiltrating lymphocyte phenotypes, and off-target immune cell activation. Secondary goals look at improving pharmacokinetic properties that limit bsApt clinical translatability while long-term goals look to generalize our findings on these properties to current and future bispecific therapies that target a wide range of solid and hematological cancers.

NIH Research Projects · FY 2025 · 2023-08

Overall Project Summary Resources and Workforce Development for Research on NIH/NIAID High Priority Pathogens at the University of Missouri Regional Biocontainment Laboratory The University of Missouri Regional Biocontainment Laboratory (RBL) was commissioned in 2009 as an $18M, 12,377 NSF BSL-3/ABSL-3 facility. During its thirteen years of operation, the RBL, since named the MU Laboratory for Infectious Disease Research (LIDR) has been a centerpiece of the broader infectious disease research community at the University of Missouri, receiving strong institutional commitment in the recruitment and sustainment of world-class faculty for management of the RBL/LIDR and for conducting research on high priority pathogens. The LIDR operates shared research resources, including state-of-the-art equipment and professional services in microbiology, aerobiology, immunology, and animal model core facilities that serve the needs of researchers on campus and in the broader regional and national communities. The faculty and professional staff of the LIDR are part of the NIH/NIAID RBL-NBL network, with collaborative interactions that facilitate sharing of best practices and knowledge, providing synergy in achieving our collective biodefense and emerging infectious disease research agenda. During the COVID pandemic, the MU LIDR, along with its RBL and NBL partners, led the response to the pandemic and are committed to strengthening pandemic preparedness of the nation, by providing BSL-3/ABSL-3 training, professional staff, and research services that allow for rapid responses in all areas, including development of animal models and the evaluation of numerous treatments, disinfectants, and vaccine platforms to combat the pandemic. The long-term goal of the present application is to sustain a leading effort for development of novel medical countermeasures for combating the ever present and changing threats of emerging and re-emerging infectious diseases, while training an outstanding cadre of next generation scientists and professionals in biocontainment. The goals of the proposed cores in Facility Management, Maintenance and Operations, BSL3 Practices, and Biocontainment Research Support Services are to educate and train the next generation of scientists and biocontainment professionals in biodefense and emerging infectious diseases, and to facilitate and enhance the development of novel approaches for prevention and treatment of infections caused by high consequence pathogens whose natural or deliberate transmission pose a threat to public health and national security. Towards these goals, the proposed Cores will work within the broader infectious disease community at MU and the NIH/NIAID RBL-NBL network for enhancement of research productivity and discovery, and continued sharing of information and best practices in order to meet the changing priorities and needs of NIH/NIAID for biodefense and emerging infectious diseases.

NIH Research Projects · FY 2026 · 2023-08

Project summary Egg generation entails a unique cell division, Meiosis I (a process also known as oocyte maturation), during which the oocyte acquires its developmental competence. For poorly understood reasons, mammalian oocytes (including human, bovine and porcine oocytes) are notoriously prone to reduced quality. Because oocyte quality is a major determinant of embryo developmental competence and pregnancy success, the long-term research goal of this application is to dissect basic molecular mechanisms that regulate meiosis I in human, bovine and porcine oocytes to understand why female gametes are notoriously prone to reduced quality. Work from our group has demonstrated that preferential initial nucleus and spindle positioning at the center of mouse oocytes is an insurance mechanism to avoid the premature exposure of the spindle to the cortical signaling that hinders proper chromosome-microtubule attachments, thereby protecting against aneuploidy, the major genetic cause of infertility and congenital defects. These findings implicate initial peripheral nucleus and spindle positioning as a major risk factor for aneuploidy in mice. Strikingly, in contrast to mouse oocytes, the majority of human, bovine and porcine oocytes have a peripheral nucleus and, subsequently, peripheral spindle formation. Interestingly, in human oocytes, peripheral nucleus positioning correlates with poor maturation rates. How the nucleus/spindle behaves when it is initially positioned peripherally in those species (humans, pigs and cattle) and whether this behavior correlates with aneuploidy are unknown. This proposal builds on these findings via two complementary albeit independent aims: (1) determine the underlying molecular mechanisms regulating nucleus behavior in human, bovine and porcine oocytes, and (2) determine whether nucleus/spindle positioning relates to aneuploidy. To carry out these aims, we will employ super-resolution microscopy, multiphoton laser ablation, genetic, optochemical and integrative multiomic approaches. These conceptually and technically innovative aims are expected to have a broad impact on the field by filling a substantial gap in our knowledge of how the nucleus/spindle behaves in human, bovine and porcine oocytes to ensure the development of good-quality gametes with clear clinical implications for assisted reproductive technologies. Such understanding is relevant to human and animal reproductive health because mammalian oocytes are notoriously associated with poor developmental competence and aneuploidy, major causes of early pregnancy loss (a high priority area of the Fertility and Infertility Branch of the NICHD).

NIH Research Projects · FY 2026 · 2023-08

Project Summary Endometrial hyperplasia is a precursor to endometrial cancer (EC). Complex atypical hyperplasia (CAH) is the common type of endometrial hyperplasia that becomes EC in 52% of cases if not treated. Most women with CAH can be cured by hysterectomy, the surgical removal of the uterus. However, there is an increasing demand for fertility-sparing treatments for CAH and EC, especially for reproductive-aged women who wish to maintain fertility. Twenty to thirty percent of the young women with CAH and EC might be eligible for a fertility sparing approach. Developing fertility-sparing treatments to cure CAH and EC without sacrificing fertility remains an essential goal in CAH and EC medicine. Poor understanding of the mechanism of progesterone (P4) resistance in CAH and EC is a major barrier to developing fertility-sparing treatment. P4 is widely used to treat various gynecological conditions due to its clear antiproliferative effects on E2-mediated endometrial proliferation. P4, the gold standard of nonsurgical treatment, is often an effective CAH and EC treatment. However, the response rates to P4 therapy vary and molecular mechanisms behind de novo or acquired P4 resistance are poorly understood. To increase success rates of P4 therapy as a fertility-sparing treatment, revealing the mechanisms underlying P4 resistance in CAH and EC and finding biomarkers for P4 responsiveness in human CAH and EC are critical. The mitogen-inducible gene 6 (MIG-6) is a key P4 signaling mediator in the human and mouse uterus. Preliminary results show that P4-responsive (Sprr2fcre/+Mig-6f/f; Mig-6Ep-KO) and P4-resistant (Pgrcre/+Mig-6f/f; Mig-6KO) mouse models develop CAH via aberrant phosphorylation of AKT and ERK in endometrial epithelial cells. In P4-responsive mice, P4 controls CAH, restores uterine receptivity, and preserves fertility. In P4-resistant mice, P4 fails to control CAH, fails to restore uterine receptivity, and fails to preserve fertility. These data suggest the hypothesis that Mig-6 loss causes P4-resistant CAH by activating AKT signaling in endometrial epithelial cells and by dysregulating P4 signaling in endometrial stromal and epithelial cells. This project will investigate the mechanism of P4-resistance by: 1) dissecting the role of MIG-6 in the interaction between AKT and PGR signaling in endometrial epithelial cells; 2) studying the function of stromal MIG-6 in response to P4; 3) testing whether combination therapy of P4 + AKT or mTOR inhibition can treat P4-resistant CAH and restore endometrial function, including fertility; and 4) conducting bioinformatic analysis study that will identify the transcriptional regulatory function of PGR and find the biomarkers in P4 resistance. This work will lead to translational outcomes including the development of new therapeutic approaches for fertility-sparing treatment as well as discovery of new biomarkers, which are important for Precision Medicine in infertility.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Unlike most other organs and tissues, the endometrium of the adult uterus has a remarkable regenerative ability, undergoing repetitive cycles of proliferation, differentiation, breakdown, and regeneration. The endometrium is a complex tissue comprised of stroma, vasculature and immune cells, as well as two major epithelial cell types — luminal (LE) and glandular (GE) epithelium. Notably, the endometrium repairs after menstruation, injury, and childbirth without scarring and then regenerates with full function to support pregnancy. Aberrations in regeneration negatively impacts pregnancy success and can lead to infertility or diseases, such as endometriosis, endometrial fibroids, Asherman’s syndrome, and endometrial cancer. Thus, the long-term research objective is to define the critical intrinsic and extrinsic mechanisms governing uterine epithelial cell differentiation and regeneration with subsequent impacts to diagnose, treat, and prevent infertility and endometrial disease in women. The regenerative capacity and ability to grow ectopically (endometriosis) suggests that the endometrium has a robust and plastic progenitor population. Indeed, numerous reports have provided evidence that cells with stem cell-like qualities exist in the epithelium of the uterus; however, the identity, behavior, and mechanisms underlying the fate of those cells and their location remains unclear. Ambiguity within the uterine stem cell field may be partly because the strict lineage hierarchies that characterize development and homeostatic tissue turnover are not followed during tissue repair. Recent studies in several organs found that epithelial plasticity and activation of facultative stem cells are common strategies for tissue regeneration in the injury repair process. Therefore, this proposal focuses more on the process used by the uterus to replace lost cells, rather than on the physical entity of a stem cell. The overarching hypothesis is that the uterine epithelium contains cells that are unipotent during normal homeostatic turnover but have the ability to dedifferentiate upon injury to coordinate successful epithelial regeneration. Guided by strong preliminary data and the use of innovative mouse genetic models, organoid culture, and single-cell sequencing technologies, two specific aims will begin testing that hypothesis: (1) epithelial plasticity in the regenerating uterus; and (2) cellular and molecular aspects of LE response to GE ablation. The outcome of the proposed studies will connect epithelial regeneration responses to specific molecular mechanisms of epithelial differentiation and repair. In the long term, an increased understanding of the cellular and molecular mechanisms that govern endometrial epithelial cell differentiation and regeneration is important not only for gaining fundamental knowledge of tissue and stem cell biology but also for the development of therapeutics for the treatment of endometrial diseases.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT: The overall goal of this research program is to delineate the role of germline epigenetic alterations (epimutations) in the onset and progression of inter-and transgenerational reproductive defects. So far, published scientific literature suggests that environmentally induced epigenetic alterations, mainly DNA methylation, histone modifications, and RNA modifications (epitranscriptome), are transmitted to subsequent generations via germline (eggs or sperm)1. However, the role of observed epimutations in the development of reproductive phenotypes is not well understood because of a lack of clear understanding of PGC to germline and germline-to soma transfer of reprogramming-resistant epimutations during the turnover of generations. Here in the proposed study, we are asking three big questions: a) Do germline epimutations establish age and developmental stage specifically in males? b) Do ancestrally established transgenerational epimutations predispose future generations to increased risks for reproductive impairment if exposed again to emerging environmental chemicals of concern? c) Is the role of the observed epimutations in the development of progression of phenotypic traits causative or correlative? This R01 research project will systematically answer these questions in three different aims. BPA will be used as a ubiquitous model endocrine-disrupting chemical (EDC), and Bisphenol S (BPS) will be used as an emerging contaminant of concern, and medaka fish as a comparative vertebrate animal model. Aim 1 will test the hypothesis that male germ cells at all stages of development are susceptible to BPA, the model EDC. Aim 2 will test the hypothesis that exposure of the F3 generation offspring with pre-existing epimutations to emerging contaminants will result in an increased incidence of reproductive impairment. Aim 3 will test the hypothesis that the EDC-induced epimutations are associated with reduced fertility in males and females and that these epimutations can be corrected by reprogrammable CRISPR-dCas9 epigenome editing in vivo. The project will identify footprints of the past exposure, determine the role of epimutations in reproductive impairment, determine whether inherited epimutations predispose current and future generations to increased risks of reproductive health due to exposure to emerging contaminants of concern, and provide significant insights into mechanisms underlying germline transmission of the epigenome and longitudinal health risks of exposure. Understanding of key time points during which epimutations transfer would provide insights into strategies for the mitigation of future estrogenic chemical-induced reproductive health effects in humans.

NIH Research Projects · FY 2025 · 2023-07

Project Description Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene. CRISPR/Cas9 editing holds promise to treat DMD at its genetic root. Since DMD affects all muscles in the body, effective therapy for DMD would require bodywide muscle delivery. Adeno-associated virus (AAV) vector is the only delivery system that can efficiently reach all body muscles. For this reason, AAV has been the vector of choice for CRISPR-mediated gene repair therapy for DMD. The AAV vector leads to persistent transgene expression. Continuous Cas9 expression creates two problems. First, it increases the odds of off-target editing. Second, the cytotoxic T lymphocyte (CTL) response to the bacterial-derived Cas9 protein can eliminate the treated cells and abolish the therapy. Many approaches have been developed to monitor off-target editing and improve gene editing fidelity. However, it has been elusive to model the Cas9-specific CTL response. Mouse studies revealed a limited cellular response that failed to eliminate Cas9 transduced cells. In fact, we and others have observed nearly lifelong Cas9 expression, muscle pathology amelioration, and function improvement in mdx mice, the most used mouse DMD model. In contrast to the mouse model, dystrophic canines are considered better models for informing human trials. To determine whether Cas9 immunity is a hurdle for AAV-mediated DMD CRISPR therapy, we performed a comprehensive study in four independent canine models (normal canines and three different canine DMD models) using both Cas9 and non-Cas9 AAV vectors via local and systemic delivery. We found compelling evidence suggesting that the Cas9, but not non-Cas9, AAV vector induced a robust CTL response and eliminated gene-edited dystrophin-positive myofibers. Our studies established a reliable model system to study Cas9 immunity. Importantly, it opens the door to developing and validating strategies that may mitigate the Cas9-specific CTL response in a clinically relevant large animal model. In this proposal, we will leverage our findings to explore novel strategies that may support persistent therapeutic editing without inducing the CTL response in the canine DMD model. Our studies will pave the way for translating AAV CRISPR therapy to DMD patients in the future. Our findings will also inform the translation of AAV CRISPR therapy for other human diseases.

NIH Research Projects · FY 2026 · 2023-07

Project Summary This research seeks to understand the fundamental process of protein translocation across membrane barriers in bacteria. To establish an infection or exchange antibiotic resistance genes, bacteria must transport macromolecules including protein across multiple membrane barriers: their own and the host cell’s. In response to the universal requirement for macromolecule export, bacteria have evolved elaborate machineries called secretion systems that use energy to move macromolecules from the bacterial cell out into the extracellular milieu or directly into a host cell. In this proposal, I focus on the Type IV secretion system (T4SS). This family of secretion systems is unique in that there are T4SSs that can transport nucleic acid and/or protein cargo. As an important example of a T4SS, I will first investigate the defect in organelle trafficking / intracellular multiplication (Dot/Icm) T4SS in Legionella pneumophila. This system is essential for pathogenesis, which can result in the potentially fatal pneumonia Legionnaires’ Disease. The Dot/Icm T4SS is composed of 30 proteins in different copy numbers. It secretes over 300 protein substrates in order to evade the host cell’s immune system and scavenge nutrients. This represents a much larger repertoire of substrates than observed in other secretion systems that transport proteins out of the bacterial cell. Thus, the Dot/Icm T4SS is an ideal model system for determining how these numerous substrates are engaged and transported. Protein transport by T4SSs has traditionally been studied using cell-based assays. The cellular environment, however, does not allow for precise control and manipulation of reaction conditions. The field needs rigorous biophysical assays with which to dissect the molecular mechanism of protein translocation. I propose to combine determination of high-resolution structures of the Dot/Icm T4SS by cryoEM and thermodynamics and enzyme kinetics studies of the system. These approaches will complement traditional genetic and cell biological strategies and will lead to mechanistic insights into how this secretion system transports protein. For example, transient state kinetics experiments observing the ATP-dependent translocation of a fluorescently labeled substrate protein will answer questions such as “which signal sequences are recognized by which motor protein(s),” “are protein substrates unfolded during transport,” and “which kinetic steps are coupled to ATP binding and hydrolysis?” This approach to investigating complex cellular machinery by integrating biochemical, biophysical, structural, and genetic approaches will shed new light on the fundamental process of translocation across multiple membranes, an important feature of bacterial pathogenesis. While this work aims to understand the fundamental mechanism of protein translocation, our findings could lay the foundation for scientists to develop anti-virulence drugs, the next generation of tools fighting bacterial disease, and to engineer the targeted delivery of gene and protein therapeutics to eukaryotic cells by secretion systems.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Genetically engineered (GE) animal models are essential for generating biomedical models for human disease and for gaining a better understanding of animal biology. Targeted modification of the animal genome allows the animals to present human disease phenotypes, and therefore, are critical to design and develop novel treatments. The use of GE large animal models such as pigs often results in clinically relevant outcomes as their physiology and anatomy resemble humans. For example, introducing genetic elements responsible for cystic fibrosis and immunodeficiency to the pig genome induces GE pigs to closely recapitulate symptoms of the diseases. However, GE pigs are not widely available in biomedicine due to the amount of time required to establish such models. As a large animal species, a single round of breeding in pigs takes at least one year and often multiple rounds of breeding is necessary to establish GE pig models. Application of genome editing tools, such as the CRISPR/Cas system, has significantly improved efficacy to introduce targeted modifications to the pig genome. However, concerns over unintended genome alterations from genome editing procedure and days required to introduce targeted modifications in pigs as a large animal model impedes wide use of the technology. Our objective of this project is to evaluate the efficacy and safety of genome editing technology and design novel approaches that will assist in rapid phenotyping of animal models after a targeted genome editing event. Three specific aims are proposed to reach our goal. First, we will generate methods for global detection of off-targeting events in GE pigs. Secondly, we will develop strategies to secure genome integrity during the genome editing process. Finally, we propose to develop a strategy to rapidly phenotype GE fetuses and to modify the genome of wild-type pigs. Targets of this third aim are genes associated with traits that are relevant in both agriculture and biomedicine. The knowledge obtained from this project can be implemented to expand the use of GE pigs in biomedicine while also having an impact on agriculture production. The importance of using the genome to predict the phenotype for rapid identification of improved alleles and traits will be grown here. Our expertise in using genome editing technology and GE pig models will be employed to complete the proposed aims. Outcomes of this project should increase the availability of GE pig models in biomedicine and agriculture by effectively capturing subsequent phenotypes after genome editing events. We propose to utilize pigs as a model to investigate the efficacy of the proposed strategies; however, our findings should be easily transferred to producing other animal models in biomedicine and agriculturally important species, as well. Given the importance of pigs used as animal models, our findings should be beneficial to both NIH and USDA agencies.

NIH Research Projects · FY 2026 · 2023-06

Abstract Obstructive sleep apnea (OSA) is a prevalent condition worldwide, especially in people with obesity, and is an independent risk factor for cardiovascular disease (CVD), including coronary microvascular dysfunction (CMD). Current available OSA treatments have not consistently detected the anticipated improvements in CVD and CMD, suggesting the need for adjuvant therapies aimed at the mechanisms underlying the core disturbances induced by OSA. As such, OSA-induced renin-angiotensin-aldosterone system (RAAS) activation may trigger excessive mineralocorticoid receptor (MR) signaling, which play a major role in endothelial dysfunction and atherosclerosis. Thus, the hypothesis is that OSA-induced CMD is mediated, at least in part, by MR dependent mechanisms. To examine the role of MR in OSA-induced CMD, obese C57Bl/6 male and female mice will be exposed to intermittent hypoxia (IH) during the rest period, a mouse model of OSA, for 6 weeks (short-term) and 16 weeks (long-term) and treated with the conventional steroidal MR antagonist (spironolactone) or the novel non-steroidal MR antagonist (finerenone) to evaluate the MR-dependent reversibility of IH-induced CMD (SA1). To investigate whether MR inhibition can accelerate CMD recovery, mice will be exposed to short-and long-term IH followed by 12 weeks of normoxia (IH cessation – simulating ideal OSA treatment) with or without concurrent treatment with MR antagonists (SA2). To examine the role of vascular cell-specific MR, transgenic mice with endothelial cell (EC)-specific and smooth muscle cell (SMC)-specific deletions of MR will be exposed to long- term IH (SA3). Coronary artery function will be evaluated in vivo and ex vivo in addition to heart function, blood pressure and metabolic assessments. Moreover, immunohistological analysis of the coronary vessels, along with gene networks expression dynamics among different coronary artery cell populations will be evaluated using single-nucleus RNA sequencing (snRNA-seq). Male and female mice will be fed a high-fat or control diet for 8 weeks then housed in environmental chambers for IH exposures (alternating 6.1% FIO2/21.0%FIO2 90 sec:90 sec, for 6 or 16 weeks during 12 daylight hours, and treated with spironolactone (20mg/kg), finerenone (1mg/kg), or placebo. IH cessation protocol consists of removing the exposed animals for the IH chambers and left under normoxic conditions for 12 weeks. Heart function will be examined via echocardiography, while blood pressure and coronary flow reserve velocity (CFVR) will be assessed using tail cuff method/telemetry and doppler flow velocity system, respectively. Additionally, Insulin tolerance test and fasting blood and lipid profiles will be evaluated. After euthanasia, coronary arteries will be excised and mounted on a wire myograph for functional studies or processed for immunohistological analyses (including intima-media thickness, collagen fiber distribution, and indices of oxidative stress and inflammation) or snRNA-seq. The proposed studies will elucidate the role of MR signaling in OSA-mediated coronary artery dysfunction and potential approaches to enhance CMD reversibility, thereby enabling MR antagonists as biologically plausible therapeutic targets in OSA patients.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Most human health traits are highly complex and hierarchical, in which a high-level trait (e.g., energy expenditure) is the product of the combined effects of a suite of sub-phenotypes. These trait hierarchies are typically influenced by many genetic variants of small effect that interact with one another and with environmental factors, making the identification of the causative variants a significant challenge. As a result, past research strategies have largely failed to fully address the complexity of most complex traits, leaving a significant knowledge gap in our understanding of the genetic basis of these traits. This project will use an innovative combination of a large multiparent mapping population and experimental evolution using the powerful fruit fly model system to identify the common mechanistic connections between complex traits and observe how these connections influence multitrait evolution. First, this project will simultaneously evolve flies targeting multiple trait hierarchies and track the genomic and phenotypic changes that occur during adaptation. Second, this project will leverage a large multiparent mapping population that has been used broadly in the genetics community to address fundamental questions about the generality of emergent properties of the genome, such as the extent of pleiotropy, genotype by environment interactions, and genetic background effects. Overall, this research will provide generalizable lessons about how genomes are connected to physiology to produce the interconnected set of traits that affect health across the lifespan.

NIH Research Projects · FY 2026 · 2023-04

Summary Despite technological advancements in head and neck squamous cell carcinoma (HNSCC) radiotherapies, collateral damage to surrounding normal tissues such as salivary glands following ionizing radiation (IR) remains a significant problem for these patients and severely diminishes their quality of life. It is estimated that >95% of HNSCC patients exhibit xerostomia and salivary gland hypofunction following the irradiation regimen and >73% of these patients continue to suffer for months to years after completion of radiotherapy. The lack of treatment options to prevent IR-induced xerostomia or recover salivary function is compounded by a limited understanding of the underlying mechanisms that mediate chronic IR-induced salivary dysfunction. Our previous studies demonstrate the action of extracellular ATP (eATP), a key “alarmin” molecule in damaged tissue that contributes to IR-induced salivary dysfunction, can be inhibited by pharmacological or genetic blockade of P2X7Rs for eATP, thereby conferring significant radioprotection to salivary glands. In addition, our studies show that radioprotection from P2X7R antagonism maintains normal salivary output through day 30 post-IR, although longer time periods relevant to the treatment of IR-induced chronic xerostomia and salivary dysfunction have not been investigated. Our ultimate goal is to use P2X7R antagonists as radioprotective agents to retain salivary gland function in head and neck cancer patients undergoing radiotherapy, although there is little information available on whether P2X7R antagonists will interfere with tumor regression achieved by radiotherapy, and proposed studies will address this gap in knowledge. The overall goal of this proposal is to develop an approach using P2X7R antagonism to prevent IR-induced salivary dysfunction in head and neck cancer patients receiving radiotherapy without inhibiting the IR-induced regression of HNSCC tumors. Successful completion of this proposal will greatly accelerate translation of this novel pharmacological approach to human patients undergoing IR therapies for head and neck cancer. The following specific aims will be pursued: Specific Aim 1 will develop a radioprotective approach using P2X7R antagonism in an irradiated syngeneic mouse model of HNSCC that preserves salivary gland function and anti-tumor responses. Specific Aim 2 will investigate the durability of the radioprotective effects of P2X7R antagonism towards developing an approach for chronic salivary dysfunction that occurs in HNSCC patients following radiotherapy. Specific Aim 3 will evaluate the contribution of eATP release and purinomic signaling downstream of P2X7R activation to IR-induced salivary gland dysfunction in mouse and human salivary gland organotypic cultures towards identifying alternative radioprotective targets and translating findings in mice to humans.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Cancer cells undergo metabolic reprogramming in order to meet elevated energy requirements to fuel proliferation, thus resulting in their differential utilization of many essential metabolites compared to normal cells. Recent advancements in the field of cancer metabolic reprogramming demonstrated significant increase in efficiency of standard cancer treatments when combined with cancer metabolic inhibitors. However, tumor metabolic reprogramming remains poorly understood for the majority of cancers. Moreover, many recent reports revealed evidence that the metabolism of cancer cells in vitro can differ significantly from that of in vivo because in vitro models lack complexity of the tumor microenvironment. However, the progress of studying tumor metabolism in vivo is significantly hampered by the lack of efficient tools that allow real-time noninvasive imaging and quantification of metabolite absorption in animal models of cancer which closely reflect human pathologies. Current strategies have significant limitations and mostly rely on MRI, nuclear imaging techniques such as PET/SPECT, and endpoint ex vivo quantification of metabolite absorption (ex. MS). Here, we propose to develop a novel optical imaging platform that has several important advantages over the existing methods, and allows noninvasive evaluation of the uptake of several essential metabolites using highly sensitive and quantifiable bioluminescent imaging. The method is independent of radioactive and/or short-lived isotopes, less costly, and allows longitudinal monitoring of metabolite absorption during disease progression (e.g., cancer development or clinical intervention such as chemotherapy). While the first application of this approach has been already successfully validated by us using glucose as an example (Maric et.al., Nat Methods, 2019), we propose to expand this technology to develop novel probes to study uptake of several amino acids, fatty acids, and nucleosides that all play central role in cancer metabolic reprogramming. We will perform thorough validation of this platform in cells, healthy transgenic mice and murine animal cancer models to assure that the reagents fulfill the requirements for physiological behavior, stability, safety, and robust signal generation both in vitro and in vivo. In addition, we will optimize in vivo delivery routes, vehicles, and concentrations to achieve high signal/background ratios. In summary, the overall goal of this study is to generate a novel optical imaging platform that would become a universal analytical tool for monitoring nutrient uptake in live cells and animal models of disease. While we plan to apply this platform to unravel tumor metabolic reprogramming, the same method could be adapted for studies of several other important human pathologies, in which changes in metabolism are known to play a significant role, such as diabetes, neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), and many others. Therefore, this novel technology is expected to have a strong, enabling, and long-lasting impact on many physiological and pathological investigations in the field of metabolism and will become a valuable tool for drug discovery, applicable to oncology and other metabolic disorders.

NIH Research Projects · FY 2026 · 2023-01

Maintaining the balance of excitatory glutamate and inhibitory GABA signaling is critical for homeostasis and generating proper reflexes in response to hypoxia. Our previous studies established glutamate signaling in the nucleus tractus solitarii (nTS), the first central site for carotid body sensory integration, is exaggerated after chronic intermittent hypoxia (CIH). However, the specific contribution of GABA, which counter-balances glutamate signaling, in the exaggerated excitation is unknown. Our current goal is to address this knowledge gap and determine the extent that inhibitory GABA signaling contributes to overexcitation of the nTS after CIH. GABA signaling is controlled or modulated by GABA release, receptor (GABARs) activation, the chloride (Cl-) equilibrium potential that is set by Cl- co-transporters (NKCC1 and KCC2), and astrocytic GABA uptake via transporters (GATs). Given the present literature and our supporting preliminary data, our overarching hypothesis is that CIH shifts nTS activity to an overexcited state due to attenuated GABA signaling via reduced GABA inhibition and increased astrocyte GAT activity. Reduced GABA tips the balance of Glu and GABA signaling, and their influence on each other (i.e., cross-talk), towards greater excitation to ultimately increase chemoreflex responses. Aim 1 will determine the extent GABA signaling is altered in CIH to increase nTS excitability. Working hypothesis: Reduced GABA inhibition increases nTS excitability and cardiorespiratory function in CIH. GABA inhibition is attenuated in CIH due to reduced GABA release or GABARs on Glu neurons, altered Cl- transport and/or augmented astrocyte GAT. Aim 2 will define the magnitude nTS GABA and astrocyte GABA transporters influence Glu signaling in CIH to control neuronal and cardiorespiratory function. Working hypothesis: GABA-Glu balance is shifted towards excitation after CIH, in part due to altered GAT function, to ultimately to increase nTS excitability and cardiorespiratory function. In this application, we will utilize a multi-faceted, synergistic and integrative approach. We will use a range of techniques including single cell electrophysiology, live-cell imaging, DREADD cellular manipulation and molecular biology to ultimately decipher physiological function. We will also use AAV expression and Cre-technology in transgenic rats that allows specific recording and manipulation in GABA neurons from the single cell to whole animal. Each technique directly complements the other, allowing an unparalleled depth of study. Together, these techniques allow the vertical study of the system and meticulous cellular investigation. Upon completion of the proposed research, we expect to identify the significance and mechanisms of elevated nTS activity due to reduced GABA signaling that result in cardiorespiratory abnormalities in IH diseases.

NIH Research Projects · FY 2024 · 2023-01

The ability coordinate breathing, suckling, and swallow is required for survival at birth. Infants with prenatal exposure to opioids can have abnormalities in swallow efficiency and swallow-breathing coordination, sensory- motor responses to pharyngeal stimulus, and esophageal motility reflexes, which often results in persistent feeding difficulties. The swallow pattern generator is located within the brainstem, but it has been difficult to study due to its location and widely dispersed neural circuits. Our preliminary studies describe a novel method that allows for the visualization and recording of a vast number of neurons along the intermediate zone of the medial reticular formation and Nucleus Ambiguus (NA), while preserving the Nucleus Tractus Solitarius. We demonstrate reliable central stimulation of swallow with simultaneous optical recording of neurons in the semicompact and compact regions of the NA, along with nerve root recordings. Activation of NA neurons during swallow is affected by prenatal opioid exposure. Our methods also allow superimposition of optical recordings onto subsequent immunohistochemical images to identify regional cellular phenotypes and to confirm recording locations. Our working hypothesis is that the brainstem swallow network spans the medulla and is vulnerable to opioids. The current application has evolved from collaborative work from two established investigators, bringing together expertise in central neural circuitry, optical/ histological techniques, and regulation of swallow. The proposed studies are designed to describe location and type of neurons active during swallow, alterations when swallow is stimulated across the respiratory cycle, and the impact of neonatal opioid withdrawal syndrome. The combined state-of-the-art techniques will provide much-needed mechanistic insight into swallow and clinical observation of infants with neonatal opioid withdrawal syndrome (NOWS).

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Vaccination is the greatest public health achievement of our time. With an explosion of antibiotic resistance, new vaccines against multi-drug resistant (MDR) bacterial pathogens are more important than ever. Pseudomonas aeruginosa (Pa) is an opportunistic human pathogen that causes severe infections in patients with cystic fibrosis (CF), burns, severe wounds, pneumonia, as well as critically ill patients who require intubation or catheterization. Clearing Pa has become problematic as it has become increasingly antibiotic resistant. This is exacerbated by the fact that the biggest risk factor for negative outcomes associated with MDR Pa is advanced age. After 60, there is a significant increase in morbidity and mortality resulting from MDR Pa. While there are Pa vaccines in development, none are licensed. Like many Gram-negative pathogens, Pa strains of the PAO1/PA14-clades possess a type III secretion system (T3SS), a virulence factor that allows avoidance of host innate immunity and is required for the onset of infection. Structurally resembling a molecular syringe with an external needle, the T3SS apparatus (T3SA) provides an energized conduit from the bacterial cytoplasm into the host cell for transporting effector proteins that mediate key aspects of infection. A needle tip protein and the first of two translocator proteins localize to the distal end of the T3SA needle to mediate host cell contact. These proteins, PcrV and PopB, respectively, are required for pathogenesis and are 95-98% conserved among Pa. We have fused PcrV and PopB to give PaF. After demonstrating the protective efficacy of PaF, we genetically fused LTA1, the active moiety of labile toxin from ETEC, to the N-terminus of PaF (L-PaF). L-PaF reduces mouse and rat lung burdens significantly. When compared to PBS-vaccinated mice, L-PaF-vaccinated mice possessed significantly higher OPK activity in the sera and elevated levels of IL-17 were secreted from lung cells. Recently, Pa outliers have been identified that are devoid of the T3SS entirely and use ExlA to disrupt host cell membranes. Thus, we have added ExlA to our L-PaF (L-PaFE) emulsion and have demonstrated protection in PAO1/14/7 clades when delivered intranasally. Furthermore, we have added BECC438, a novel monophosphoryl lipid A (MPL) biosimilar (a TLR4 agonist), to increase OPK activity (L-PaFEB438). The goal of the R01 is to continue to develop our broadly protective Pa vaccine formulation by assessing the protective immune response in rodent models. Knowing the vaccine efficacy and immune response in these models will allow us to finalize the vaccine formulation and the demonstrate the potential utility of that formulation in humans.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Nanomedicine provides new opportunities to solve medical problems that were previously perceived as unsolvable by clinicians. One such problem is drug resistance in Non-Small Cell Lung Cancer (NSCLC). Attempts to overcome the resistance using small molecule inhibitors have failed to restore the drug sensitivity. Alternative compensation survival pathways emerge to allow the regrowth of the tumor. To abate the tumor growth, we need to suppress more than one survival pathway with minimal or no side effects. Nanoparticles possess the ability to selectively and simultaneously arrest multiple survival pathways to control the growth of the tumor. However, nanoparticles to co-knockdown multiple cross-linked survival pathways have not been explored yet. We recently identified two targets AXL and FN14 and demonstrated that the cross-talk between these markers is responsible for resistance in EGFR mutant NSCLC. Subsequently, we designed nanoparticles to simultaneously co-knockdown both AXL and FN14 in the tumor, disrupted the cross-talk, and successfully overcame drug resistance in murine models. The logical next step is to validate the findings in clinically relevant animal models and patient samples. It is equally important to optimize the size of nanoparticles further to maximize deep tumor delivery with favorable in vivo characteristics. Therefore, in this proposal, we will synthesize different sizes of NP, understand their ability to penetrate deep inside the tumor to fully restore drug sensitivity for a long-lasting therapeutic response. We will use patient tissues, patient-derived organoids, drug- resistant cell lines, orthotopic animal models, and patient-derived xenografts to establish the efficacy of the nanoplatform. The data will validate our new nanoparticle platform, as a promising strategy to combat drug resistance in NSCLC and catalyze clinical trials in the future.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY / ABSTRACT The overarching goal of this study is to use new large multi-modal data resources and machine-learning-based data mining algorithm to better understand risk factors and improve diagnosis for people with Amyotrophic lateral sclerosis (ALS). Amyotrophic lateral sclerosis (ALS) is a rare, fatal neurodegenerative disorder, with 90% sporadic cases do not have genetic causes and their contributing risk factors are largely unknown. Most of what is known about ALS risk factors comes from epidemiological studies using registry data, which historically forms the main standardized big data source to help describe the natural history, epidemiology, and burden of disease; however, the strength of evidence resulting from these studies varies greatly. One potential major limitation to registry data are the fields collected are based upon known potential risk factors, which have restricted its usability for exploring novel associations and causalities. Moreover, ALS is a rare disease with low prevalence, thus making it infeasible to study its etiology using traditional observational study design due to statistical power constraints. The digitization of healthcare records and the capacity to link to other relevant data sources now enables a more representative, enriched and statistically powerful study population; and ideal for leveraging machine-learning-driven, hypothesis-generating models to identify new risk factors and patterns identify new risk factors important for understanding, diagnosing, or treating people with ALS. Building on established well-integrated real world big data source and established ensemble embedded feature selection framework, an established multi-marker (biomarker, clinical marker, geo-marker, socio-marker) discovery algorithm will be developed to discover novel, generalizable risk factors (Aim 1); new symptomatic patterns for early diagnosis (Aim 2), and effective clinical care pathways for ALS (Aim 3). To best translate findings into clinical insights, a multi-disciplinary and multi-stakeholder team has been assembled, including not only investigators with diverse expertise in statistics, machine learning, clinical research informatics, neurology, computer science, epidemiology, but also an engaging patient advisory board with diverse social background. The proposed work will be one of the first pilot studies applying AI/ML-based, hypothesis-generating algorithms on statistically powerful real-world data to bridge the knowledge gap on ALS risk factors. The work will not only provide CDC agency of toxic substance and disease registry (ATSDR) with empirical evidence to better prioritize future decisions on expanding the ALS registry risk factor survey but serve to inform better designed proposals for future etiological studies and targeted trials for ALS. This study will also provide an exemplar framework which can be generalizable to advance research of other rare and complex disease domains by leveraging real world evidence.

NIH Research Projects · FY 2026 · 2022-09

Endometrial cancer is the most common gynecologic malignancy, with an estimated 66,570 new cases in 2021. Although early-stage and low grade endometrial cancer generally exhibits a favorable prognosis, metastatic and recurrent endometrial cancer is incurable with currently available standard therapies for most women. Therefore, there is an urgent obligation to explore the mechanism of tumor metastasis and recurrence to further elucidate the progression of endometrial cancer. We have developed a genetically engineered mouse model for metastatic and recurrent endometrial cancer that implicates coexistent Pten and Mig-6 mutations in endometrial cancer. Pten mutation is not sufficient for distant metastasis, but mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. After hysterectomy at stage I of endometrial cancer in mutant mice with deficiency of Pten and Mig-6, the double mutant mice developed recurrence of endometrial cancer in the abdomen and lung. Our preliminary results show that the expression of genes related to cholesterol biosynthesis pathway was significantly increased in the mutant mice. Based upon these results, we hypothesize that MIG-6 suppresses metastasis and recurrence in endometrial cancer with PTEN mutation by inhibiting cholesterol biosynthesis. Our Specific Aims are directed at understanding: 1) the tumorigenic effects of MIG-6 loss in recurrence of endometrial cancer with PTEN mutation; 2) the molecular signature of primary tumor, circulating tumor cells, and recurrent tumor in the mutant mice; and 3) the ability of statins to prevent recurrence in endometrial cancer. There is strong innovation in the novelty of our hypotheses and cutting-edge technical approaches. In particular, we will employ the first preclinical animal model that closely resembles human endometrial cancer with distant metastasis and recurrence.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Cryogenic electron microscopy (cryo-EM) has emerged as a major experimental technology to determine protein structures as it reached atomic resolution (1.2-4Å) in recent years. Compared to traditional techniques (i.e., X- ray crystallography and nuclear magnetic resonance), cryo-EM has the unique capability of determining the quaternary structures of large protein complexes and assemblies difficult or impossible for them to handle. The advance of cryo-EM technology has stimulated a revolution in structural biology of studying large protein complexes and assemblies that cannot be well studied before. However, the computational reconstruction of protein structures from cryo-EM image data is still a time-consuming, labor-intensive, error-prone, and often inaccurate process, due to the bottleneck in picking protein particles in cryo-EM images, substantial noise in 3D cryo-EM density maps generated from particle images, and lack of automated and accurate methods to build protein structures from density maps. We plan to develop advanced deep learning methods to reconstruct protein structures automatically and accurately from cryo-EM images data, leveraging the large amount of high- resolution cryo-EM data accumulated in the field and the latest advances in the deep learning technology. We will develop 2D transformer networks built on top of the attention mechanism that perform better than traditional convolutional and recurrent neural networks in image processing to pick single protein particles accurately and automatically in cryo-EM image data via a novel combination of unsupervised and supervised learning. Moreover, we formulate the problem of denoising 3D cryo-EM density maps generated from 2D particle images as a novel machine learning problem and will develop both 3D deep autoencoders and rotation- /translation-equivariant transformer networks to remove noise in cryo-EM density maps. Furthermore, we will develop end-to-end 3D rotation-/translation-equivariant networks to directly identify the backbone atoms of proteins from 3D density maps without using any known structure as template, which will be used by a novel hidden Markov model to build the high-resolution full-atom structures of any protein. The methods will be rigorously evaluated on the large amount of cryo-EM data and compared with existing methods. All these methods will be integrated together to create a fully automated machine learning pipeline, the first of its kind in the field, to reconstruct protein structures more accurately from cryo-EM images than existing methods. We will implement the individual deep learning methods as well as the entire pipeline as open-source packages released at GitHub for the community to use. We will further validate the tools and pipeline by applying them to the new cryo-EM data of a group of important membrane protein complexes (i.e., ion channels) to be generated at the Brookhaven National Laboratory.

NIH Research Projects · FY 2025 · 2022-09

Summary Sepsis is life-threatening organ dysfunction due to a dysregulated host response to microbial infection, and is a global healthcare problem with high incidence and mortality rates, responsible for 20% of deaths worldwide. In the USA, sepsis is the most common cause of death among hospitalized patients and the total hospital costs of treating sepsis are estimated at more than $24 billion annually. The high mortality rate of sepsis is due in part to delays in diagnosis and management, as a result of its initial atypical and nonspecific symptoms and lack of early and sensitive diagnostic test. The goal of this program is to develop an ultra-sensitive, highly selective, and portable solid-state nanopore sensing platform to profile a panel of sepsis protein biomarkers in clinical samples instead of one specific biomarker as currently used in the clinical setting to provide more comprehensive parameters for accurate diagnosis of sepsis at the early stage and monitoring the treatment prognosis. Aim 1: Utilize procalcitonin (PCT) as a model protein to demonstrate the feasibility of utilizing our proposed solid-state nanopore sensing strategy, which takes advantage of a combination of magnetic beads, sandwich immunoassay, DNA reporter probe cascade and amplification, and DNA-functionalized gold nanoparticles (AuNPs), as an effective generic approach for the sensitive and accurate detection of proteins in clinical samples. To optimize the sensor sensitivity, the effects of various factors such as incubation time, denature temperature, AuNPs diameter, DNA reporter probe length, nanopore dimension, etc. on PCT detection will be examined by using a silicon nitride nanopore. Furthermore, we will construct dose-response curve for PCT, and perform selectivity study & simulated serum sample analysis. Aim 2: Build on the nanopore-based PCT detection methodology developed in aim #1 to develop a nanopore-based multiplexing sensing platform for simultaneous detection and quantification of multiple sepsis protein biomarkers. An array of seven silicon nitride nanopore sensors will be constructed and used to quantitatively detect PCT, C-reactive protein (CRP), interleukin-1 (IL-1), IL-6, presepsin (soluble CD14 subtype), tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) in protein mixtures at various concentrations. Aim 3: Analyze clinical serum samples. To evaluate the effectiveness of utilizing our developed solid- state nanopore sensing platform for accurate sepsis diagnosis and prognosis, the multiplexing nanopore sensor array developed in aim 2 will be used to analyze 120 clinical serum samples from sepsis patients at different stages and healthy controls, as well as from sepsis patients who received antimicrobial therapy. The concentrations of PCT, CRP, IL-1, IL-6, presepsin, TNF-α and LPS in these serum samples will be determined. In comparison, these samples will additionally be analyzed using ELISA detection kits.

NIH Research Projects · FY 2025 · 2022-09

Six percent of women under 44 are infertile, 12% have difficulty getting pregnant or carrying pregnancies to term, and over 75% of failed pregnancies involve implantation defects. Endometriosis, afflicting more than 10% of women of reproductive age, is a major cause of infertility, but its etiology is unclear. Therefore, identification of mechanisms involved in the pathogenesis of endometriosis-related infertility are critical. We have evidence that HDAC3 attenuation is implicated in infertile women with endometriosis. Induced endometriosis animal models involving baboons and mice showed a significant reduction of HDAC3 during the progression of endometriosis. Uterine-specific Hdac3 knock-out mice were sterile due to a defect of implantation and decidualization. In this proposal, our central hypothesis is that HDAC3 maintains steroid hormone regulation for endometrial functions, and HDAC3 loss causes infertility due to defects of implantation and decidualization. Our objective is to help solve infertility and endometrial diseases by revealing how HDAC3 functions in the uterus, and how epigenetic regulation and ovarian steroid hormone signaling are correlated. First, we will determine the pathophysiological role of HDAC3. Second, we will determine the effects of HDAC3 loss on endometriosis-related infertility. The proposed study will motivate the development of preclinical drug therapies for infertility and endometriosis and provide evidence of the molecular link between HDAC3 and steroid hormone signaling in order to accelerate evaluation of an emerging therapy against early pregnancy loss.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Long-term, continuous monitoring of heart electrical activities via electrocardiogram (ECG) plays a critical role in early diagnosis and timely interventions of various heart diseases. Concurrent detections of heart mechanics via seismocardiogram (SCG) can yield important data that complement ECG with enhanced utility in early detections of cardiac complications. However, existing ambulatory cardiac monitors are often single-modality and can only detect ECG. Moreover, they usually suffer from poor long-term usability because nonporous constituent materi- als limit their user-friendliness and long-term biocompatibility. To overcome these handicaps, this project aims to develop multifunctional porous soft materials and explore their applications in next-generation user-friendly skin-interfaced cardiac patches with bimodality (concurrent ECG and SCG recording) and long-term biocompat- ibility. The central hypothesis is that rationally designed porous constituent materials and judiciously tailored device fabrication process can enable next-generation skin-interfaced wearables with outstanding user-friendli- ness-related properties (e.g., skin-like compliance, high breathability, antimicrobial) without sacrificing their elec- trical performances. Two research aims include i) developing multifunctional porous elastomer with antimicrobial property via phase separation and investigating extrusion printing of silver nanowire-based conductive materials on obtained porous elastomers; and ii) fabricating mobile cardiac monitoring system with porous materials and evaluating its performance via on-body tests. The major innovations include (1) creation of unprecedented mul- tifunctional porous soft materials that can simultaneously achieve skin-like compliance, high breathability, anti- bacterial, and waterproof; (2) establishment of maskless, additive, high-throughput fabrications of bioelectronic devices on porous materials; and (3) generation of novel skin-interfaced cardiac patch that can outperform con- ventional ones in terms of its user-friendliness, long-term biocompatibility, and long-lasting, reliable, concurrent ECG and SCG recording. From a clinical perspective, the enabling cardiac monitoring device can shift the current paradigm of ambulatory cardiac monitoring and benefit the people who suffer from heart diseases by providing unprecedented user-friendliness for patients to wear and collecting real-time, reliable, comprehensive (ECG and SCG) data for physicians to make crucial care decisions. From a fundamental science perspective, the proposed research concerns foundational questions in skin-interfaced wearables: how to improve the user-friendliness and long-term biocompatibility of skin-interfaced wearables via material innovations (e.g., development of multifunc- tional porous soft materials) and how to fabricate high-performance bioelectronics with porous materials. From a technical perspective, the created materials and addressed fabrication principles can be used to construct various customized skin-interfaced wearables with outstanding user-friendliness, long-term biocompatibility, and long-lasting fidelity of biosignals recording to meet a variety of arising requirements of home-based, personalized healthcare (e.g., monitoring of wound healing, sleep, surgical recovery, stress, COVID-19, and elderly falls).

NIH Research Projects · FY 2024 · 2022-09

Abstract Exposure of Carbofuran (CF; a pesticide) and Chlorine (Cl2; a bleaching and warfare agent) to humans is a major public health issue. Over 3 million people exposed to CF/Cl2 show high ocular morbidity. A major gap in knowledge is lack of mechanistic data “how CF/Cl2 exposure causes corneal injury and vision loss”? We test a novel hypothesis that mTORC1-mediated dysfunctional autophagosome formation and lysosomal biogenesis are a dominant operating mechanism for corneal damage from exposure of these threat chemicals (CF/Cl2). Autophagy and lysosomal biogenesis play a key role in corneal homeostasis and transparency maintenance. Our hypothesis is based on a human-patient study identifying defective autophagy the cause of slow and progressive corneal thinning and vision loss in keratoconus patients. Pilot studies performed with mice and donor human corneas strongly supports our novel hypothesis. The main goal of this project is to test our hypothesis employing highly rigorous approach using 2 threat chemicals (CF and Cl2) and 2 animal species (C57BL/6J mice and New Zealand White rabbits) and applicability of postulated mechanism in human using donor human cornea derived organ culture and primary cell culture models using two entirely independent but integrated specific aims. Aim-1 defines clinical signs and underline mechanism of late/chronic corneal toxicity caused by CF exposure to the eye using 3 sub-aims: (1a) records clinical signs and changes in the phenotype and density of corneal epithelial, stromal keratocytes, and endothelial cells in CF +/- exposed eyes of live rabbits and mice in vivo every two weeks interval with slit-lamp, HRT3-RCM confocal, Specular, and Spectralis optical coherence tomography microscopy system until 6 months, (1b) defines the mechanism by analyzing autophagosomal and lysosomal signature genes (ATGs, LC3, SQSTM1/p62, LAMP1, mTORC1, TFEB, & vATPase) in CF +/- exposed corneas of 2 species collected at 1, 2, 4, and 6 months, and (1c) verifies applicability of postulated mechanism in human using cadaver corneas. Aim-2 defines clinical symptoms and underline mechanism of late/chronic corneal toxicity caused by Cl2 exposure to the eye using 3 sub-aims: (2a) records clinical signs and changes in the phenotype and density of corneal epithelial, stromal keratocytes, and endothelial cells in Cl2 +/- eyes of live rabbits and mice in vivo, (2b) elucidates Cl2-induced corneal damage by studying autophagosomal and lysosomal signatures stated in aim-1b in Cl2 +/- corneas of 2 species, and (2c) verifies applicability of mechanism in humans using control donor human corneas employing an experimental approach and techniques stated in Aim-1a-c. With proposed studies, we expect to define start time, extent, and duration of symptoms after CF and Cl2 exposure in live animals, role of mTORC1 mediated autophagic events in CF/Cl2 exposed corneas and signature autophagosomal and lysosomal genes linked to CF/Cl2 mediated corneal toxicity. Successful completion of studies is expected to fill many knowledge gaps and advance ocular counterACT field significantly.

NIH Research Projects · FY 2026 · 2022-08

Unchanged from Original Submission Project Summary - Research There is substantive variation in preschooler’s understanding of the core number concepts (e.g., cardinality) and associated skills (e.g., counting; Geary & VanMarle 2016) that are predictive of later individual differences in readiness to learn formal math at school entry (Geary et al. 2018) and risk of math LD (Chu et al. 2019). Child-centered interventions reduce these gaps (Dumas et al. 2019) but suffer from fade out and thus do not typically confer long-term benefits (Bailey, 2019). Similar fade out is found with individual therapy with young offenders, but sustained gains can be achieved with multisystemic interventions involving the child, home environment, and school (Henggeler et al. 2009), and it is time to consider this approach for LD. The proposed project will provide a unique and much-need foundation for the development of multisystemic – including child, parents, home, and school – interventions for preschoolers who are at risk for long-term math difficulties, including math LD. The home component will provide the most thorough assessment ever conducted of the numeracy-related home environment, including assessment of parent’s math achievement and the complexity of the math-talk with their children, as related to preschoolers’ core conceptual number development (Zippert & Rittle-Johnson 2020). The classroom component will include teacher-report and direct observation of students’ engagement in learning opportunities and the complexity of the math presented in classrooms. The child- centered assessments will focus on the longitudinal development of the core number knowledge and quantitative skills that predict later school readiness for math learning and risk of math LD (Chu et al. 2019; Geary et al. 2018). This ambitious combination will enable the first-ever dynamic assessment of the multiple contextual and child-centered factors that contribute to children’s early math development, including factors that indicate risk of later math LD. Unique features include direct assessment of parents’ math competencies; assessments of child-evocative effects (e.g., does children’s number knowledge predict longitudinal change in the complexity of parent-child number talk?); and assessments of students’ engagement in preschool classrooms. In all, the focus on core quantitative knowledge and skills, combined assessments of parents, the home environment, classroom engagement and learning experiences, and child characteristics will provide the broadest and most thorough study to date of the multiple influences on early mathematical development and preparation for school entry and risk of math LD. The combination will provide the foundation for early multisystemic interventions to better prepare at-risk children for math learning.