West Virginia University

NIH Research Projects · FY 2025 · 2024-09

Abstract: The extracellular matrix (ECM) plays a pivotal role in the structural integrity and biochemical functioning of neuronal tissues. Within the retina, a specific ECM known as the interphotoreceptor matrix (IPM) interfaces photoreceptors with the retinal pigment epithelium (RPE), implying a significant role in key processes such as nutrient transport, the visual cycle, and phagocytosis of outer segments. Mutations affecting the IPM lead to blinding diseases; however, the specific role of the IPM in retina homeostasis and blinding diseases remains elusive. This project explores the function of two functionally interdependent proteoglycans, IMPG1 and IMPG2, within the IPM and how mutations in these molecules are associated with retinitis pigmentosa (RP). Specifically, we propose three comprehensive aims: AIM 1. Investigate IMPG1/2 role in the retina-RPE nutrient exchange. Using an IMPG1/2-double knockout mouse model, this aim will test how IMPG1/2 facilitates the nutrient interchange between the retina and RPE and its link to adult-onset RP. Fluorescent and 13C6-glucose probes will be used to assess the nutrient flux between RPE and photoreceptors. AIM 2. Determine the mechanisms that lead to childhood-onset RP linked to IMPG2 mutations. Employing newly developed mouse models based on human disease, the study will elucidate the role of truncated IMPG2 proteins in accelerating the onset of RP and test gene therapy strategies. AIM 3. Identify the cause of RP linked to IMPG1 mutations. This aim proposes that mutations affecting the standard proteolysis of IMPG1 lead to intracellular protein accumulation and rod degeneration. A new mouse model, IMPG1-L583P, will be employed to test this hypothesis and treatment strategies. This research project seeks to delineate the role of IPM in retina homeostasis and unravel the mechanisms of RP disease linked to IMPG1 and IMPG2 mutations. Furthermore, this project uses new mouse models based on human diseases to test therapeutic approaches. Understanding the role of ECMs on the neuron-glia interaction holds significant potential to comprehend a broad spectrum of neuronal and retinal diseases and identify novel therapeutic avenues.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT: Open fractures are frequently (more than 150,000 cases in the U.S. every year) seen and have high (~10%) infection rates. Open fracture-associated infections are clinically devastating, and have led to reduced limb function, secondary operations, delayed union/nonunion, and death. Infection has led to significantly high costs in healthcare, resulting in approximately $500 million of additional costs in managing open fractures each year in the U.S. Further complicating the problem, alarmingly high percentages (e.g., 32.2% for tibial fractures) of open fracture infections are associated with antibiotic resistant bacteria, which have led to a two-fold increase in morbidity and mortality, and have made the drug choices for infection management increasingly limited. The long-term goal of this application is to develop translational strategies to prevent or treat bone infections in clinical settings. The objective of this project is to develop safe antimicrobial nanohybrid methods to reduce antibiotic resistant infections in open fractures. The central hypothesis is that nanohybriding two unique antimicrobial materials with different dimensions at the nanometer scale will synergistically enhance antimicrobial properties and significantly reduce host toxicity. There are three specific aims: (i) To test the hypothesis that innovative silver nanoparticle-carbon nanotube (AgNP-CNT) nanohybrids present high antimicrobial properties against various bacteria seen in open fractures and low toxicity toward cells important to bone. (ii) To test the hypothesis that AgNP-CNT nanohybrids present high antimicrobial properties and low host toxicity in preventing antibiotic resistant infections in an animal model. (iii) To test the hypothesis that bioengineering AgNP-CNT nanohybrids on implant surfaces enhances preclinical outcomes. Specifically, the in vitro antimicrobial properties of AgNP-CNT nanohybrids will be tested against a variety of bacteria and their cytotoxicity properties toward multiple human cells will be determined. The nanohybrids will be tested in an open femur fracture rat model to assess their effects on infection, healing, and host toxicity. Moreover, the nanohybrids will be bioengineered on orthopaedic implants and their coatings will be tested both in vitro and in vivo; delivering antimicrobials at the implant surface is expected to achieve high antimicrobial levels at the right place to minimize systemic toxicity. This project will contribute new knowledge on how hybriding two materials with different dimensions at the nanometer scale may synergistically increase antimicrobial properties and reduce host toxicity. The expected outcome is a biologically safe, nanotechnology-based strategy that will drive and broaden the safe use of silver and carbon nanotubes for local biomedical applications (e.g., open fracture fixation). The nanohybrids may be engineered on various medical products – ranging from bone grafts, dental implants, and catheters, to bandages and needles – to reduce infections while improving recovery.

NIH Research Projects · FY 2024 · 2024-08

Asparagine-linked glycans are involved in complex regulation and signaling, and play a critical role in disease. This is because small differences in this post translational modification dramatically change the function of any biomolecule. To fully unravel and leverage the role of glycosylation in human health, a wide array of glycan standards is required to facilitate this research. There is a critical need to customize N-glycan standards, but the chemical structure of glycans makes this process challenging. This is because glycans are defined by the variation in the type of monomeric saccharide unit, the position of the linkage between adjacent saccharide monomers, and the chain branching. Even simple glycan standards are costly, and this cost increases dramatically with increasing structural complexity. The objective of this project is to bridge this technology gap by providing a tool to customize enzyme- based N-glycan remodeling through an automated nanoscale and microscale approach. A capillary electrophoresis method is adapted to trim and rebuild glycan structures within minutes using a single- capillary or 8-capillary instrument. The heart of this system is a thermally reversible nanogel that sustains and integrates enzyme conversion of a heterogeneous substrate into a standardized glycoform product. Once synthesized, these glycans are easily conjugated to any biomolecule resulting in a custom glycoform. The proposed technology is ideal for small scale processing because the small volume of the 25-100 µm inner diameter fused silica electrophoresis capillary is compatible with the 0.5 to 100 µg quantities of each glycoform needed to create an experimental test set, or even a molecular library. A major advantage to in-line enzyme reactions is that it reduces the volume of a bench top process to the nanoliter regime. By scaling down the volume of the enzyme conversion, diffusion limited processes are less significant. An attractive feature of nanogels is that an enzyme can be pseudo- immobilized within the highly viscous gel without using covalent chemistry to anchor the enzyme. This new strategy for processing of N-linked glycan structures is facilitated through two independent approaches that differ in the quantity of product that is made. Aim 1 creates a discrete stepwise modification through the successive use of trimming (exoglycosidase) and building (transferase) enzymes. This enables automated production of microgram quantities of research grade N-glycans. Aim 2 transfers and scales up the enzymatic processing using continuous feed and parallel reaction capillaries. The proposed activities are significant because the speed and automation of the electrophoresis-based foundry yield previously unattainable flexibility in chemical processing. This new tool provides the standards needed for individual researchers to obtain direct information about the relationship between complex variations in glycosylation and physiological effect.

NIH Research Projects · FY 2025 · 2024-08

Rural Appalachian youth are at disproportionately high risk for suicide and experience significant barriers to health care access. School-Based Health Centers (SBHCs) have the potential to address suicide risk among this underserved population by providing routine evidence-based suicide risk screening, assessment, and follow-up. The NIMH Clinical Pathway is an evidence-based suicide risk detection and management program that has been found to be effective at addressing suicide risk among urban youth. However, the feasibility and impact of the NIMH Clinical Pathway have not been evaluated in rural Appalachian SBHCs, who face unique contextual, resource, and cultural barriers to implementation. To address this health care disparity, we propose a pilot hybrid effectiveness-implementation type-1 study that will use mixed methods and a stepped wedge design to adapt, implement, and evaluate the NIMH Clinical Pathway in two SBHCs located in rural Appalachia. This study will gather data regarding the specific needs of rural Appalachian youth and providers in the SBHC setting, use a Quality Improvement (QI) approach to modify the pathway and tailor implementation, and gather preliminary data on the effectiveness and sustainability of the modified pathway. Data, training materials, and implementation strategies developed can be disseminated to practices throughout rural Appalachia and will be used to support a future R01 trial of the NIMH Clinical Pathway in these Appalachian settings. This study has the following three aims: Aim 1: To adapt the NIMH Clinical Pathway and tailor implementation to address the unique needs of the rural Appalachian SBHC care setting and gather preliminary data on its feasibility, appropriateness, and acceptability. Aim 2: To examine the preliminary effectiveness of the adapted NIMH Clinical Pathway compared to Treatment as Usual (TAU) on overall screening and detection rates as well as service and patient outcomes for youth identified as at risk for suicide. Hypothesis 2a: Overall proportions of screening will be significantly higher during the intervention phase than during TAU and risk detection rates will increase. Hypothesis 2b: Youth receiving the modified NIMH Clinical Pathway will show improvements in service and patient outcomes (safety planning, follow-up visits, suicidal ideation/behavior, depression, and safety plan/acute care usage) compared to TAU. Aim 3: To identify barriers to and facilitators of implementation and sustainability of the adapted NIMH Clinical Pathway in the rural Appalachian SBHC setting. This innovative pilot study will address a critical health care disparity by identifying strategies to increase the widespread adoption of an evidence-based suicide prevention program for rural Appalachian youth.

NIH Research Projects · FY 2025 · 2024-08

SUMMARY The goal of this proposal is to develop therapeutic antibodies for the treatment of multidrug resistant (MDR) P. aeruginosa infections, with an emphasis on sepsis. With the rise in antimicrobial resistance around the world we are running out of therapeutic options against MDR P. aeruginosa. Our laboratory has identified a potential solution to address this problem: a therapeutic antibody cocktail that targets the lipopolysaccharide of P. aeruginosa. One of the antibodies present in the cocktail (WVU-VDC-S3D4, or S3D4 for short) completely protects mice against lethal sepsis, preventing bacterial dissemination and cytokine storm. This antibody is also more potent than vaccination with a P. aeruginosa whole cell vaccine or passive immunization with serum from whole cell vaccinated mice. Most interestingly, S3D4 is also capable of directly killing P. aeruginosa in vitro in the absence of complement or immune cells. In this proposal, we will characterize the mechanism of action of S3D4, formulate it in an LPS multivalent antibody cocktail, and evaluate efficacy against MDR P. aeruginosa. To do this, we will evaluate host and bacterial factors involved in S3D4 function (Aims 1 and 2). We will then combine it as cocktail with three additional antibodies that target the 6 LPS serogroups that cause 87% of P. aeruginosa bloodstream infection. Efficacy in vitro and in vivo will be evaluated with MDR clinical isolates (Aim 3). We will also evaluate efficacy against P. aeruginosa biofilms. We hypothesize that a multivalent anti-LPS cocktail of antibodies, alone or in combination with standard of care antibiotics, will be efficacious for the prevention and treatment of MDR P. aeruginosa sepsis. By the completion of these studies, we anticipate to elucidate the mechanism of action of a novel class of antibodies that can directly kill P. aeruginosa in vitro, which will help with the production of additional antibodies with similar functions against other MDR organisms. We will also produce proof of concept data to support the generation antibody therapy against P. aeruginosa infections. Altogether, this project will generate important knowledge to improve the lives of patients affected by this MDR bacterium.

NIH Research Projects · FY 2026 · 2024-07

Abstract The ability of certain animals to regenerate their cells and tissues has long been a subject of interest for biologists due to its relevance to animal development and regenerative medicine. However, we still don't fully understand why some animals regenerate so well, while others cannot. From a holistic perspective, the loss of regenerative capacity in vertebrates correlates with the evolution of larger and more complex body plans as well as the loss of asexual reproduction. However, it's unclear how body size or anatomical complexity affects regenerative processes, and the role of asexual reproduction in this process is unknown. This knowledge gap centers on a fundamental principle underlying the differential regenerative properties of animals and their tissues. Hox genes are conserved transcriptional regulators key to the evolutionary diversification of body plans. Although Hox genes have been extensively studied in animals with limited regenerative capabilities, their functions in animals capable of whole-body regeneration are not well understood. A better understanding of Hox gene functions in highly regenerative organisms could provide valuable insight into the body plan features that underlie the retention and modification of regenerative abilities. As a trainee, I have discovered that Hox genes regulate asexual reproduction in planaria, a flatworm that can regenerate its entire body. This invertebrate model's potent regenerative abilities and amenability to advanced molecular analysis and large-scale gene interference screens make it ideal for studying the fundamental mechanisms of animal regeneration. Our recent preliminary data reveal a new role of Hox genes in limiting the body size of planarians. Taking advantage of this finding, we generated animals five times their normal size and discovered that planarian regenerative abilities cannot functionally scale in an expanded body plan. Finally, we have identified genes with dual functions in both whole-body regeneration and asexual reproduction from a large RNAi screen of transcriptional regulators, which lays the foundation for uncovering a shared gene regulatory network linking these processes. Through the mechanistic dissection of how Hox genes support planarian whole-body regeneration, I aim to expand our understanding of the functions of these key developmental genes while providing important insight into the fundamental principles distinguishing regenerative versus non-regenerative body plans. This work has the potential to enhance our understanding of the permissive factors underlying tissue regeneration and to inspire and inform novel approaches to improving human health.

NIH Research Projects · FY 2026 · 2024-07

Abstract/Summary Despite the nearly 4 decades of technology development for biomolecular structure analysis since the discovery of the ribozyme, researchers still grapple with an inability to characterize important intermediates and other aspects of functional nucleic acid structure. Although powerful approaches such as X-ray crystallography, NMR, and Cryo-EM can provide atomic-level detail, they are essentially blind to structural transformations, co-existing solution structures and conformational flexibility. Time-resolved NMR provides useful information about large conformation changes on long timescales; however, it is not able to detail the conformational ensemble along a folding pathway. Mass spectrometry (MS) including MS coupled with ion mobility spectrometry (IMS) are promising tools to provide additional structural detail but suffer from two disadvantages for studying functional nucleic acids. The first is a relatively low signal intensity associated with the analysis of oligonucleotides in negative ion mode. This potentially renders many important structures (e.g., low population) essentially invisible to the analysis. The second is that the ions can undergo rapid structural transformation such as compaction in the gas phase making the extraction of solution-relevant details very difficult. Overall, MS analysis requires dramatically increased sensitivity and better means to extract solution structure information to characterize important intermediates and conformational flexibility in structure establishment. Here we propose the development of a paradigm-breaking, front-end technology platform for enhanced compound ionization and improved structure resolution. The technology is built upon the new ionization technique capillary Vibrating Sharp-edge Spray Ionization (cVSSI), which is shown to provide 10-to-100-fold ion signal enhancements over state-of-the-art ESI sources in negative ion mode. This work will extend that advantage by another order of magnitude to break through the sensitivity issues associated with the characterization of functional nucleic acids. Additionally, the work will produce an integrated platform that utilizes new, rapid mixing strategies for performing hydrogen-deuterium exchange labeling kinetics as well as oligonucleotide folding kinetics. The microfluidics device will couple seamlessly with cVSSI to accomplish high sensitivity ionization as well as to permit pulsed (in-droplet and on-line) and continuous (on-line) HDX labeling. With the device it will immediately be possible to resolve co-existing structures, structural transformations, and conformer flexibility based on differences in HDX reactivity. A novel HDX kinetics modeling methodology will also be developed to allow for structure elucidation from even the shortest timescale measurements. The new technology platform will be validated with gold standard functional nucleic acids including 10-23 DNAzyme, Hairpin Ribozyme, and Group II Intron systems. The complete mapping of conformational heterogeneity and flexibilities and folding processes of these systems will open the floodgates for further work on therapeutically-important functional nucleic acids.

NIH Research Projects · FY 2024 · 2024-07

SUMMARY Vision is how most organisms perceive and respond to their environment, making the development and function of this sensory modality paramount. Visual experience is necessary to refine visual processing, a form of experience-dependent plasticity. The majority of this refinement occurs during a brief developmental window known as a critical period, when visual input can produce extensive changes. During the visual critical period, the absence of visual experience is associated with amblyopia (lazy eye) in humans and impaired visual acuity. The circuity underlying visual transformations are well established, including the cortical changes imposed by visual experience. However, subcortical circuits (e.g. the thalamus) also exhibit a critical period and visual experience driven changes. Compared to cortical plasticity the mechanisms instructing thalamic visual plasticity are poorly understood. This gap in knowledge means we have an incomplete understanding for how visual experience refines visual circuit function and processing. In established models, technical obstacles exist to address this gap, such as anatomy (thalamus is a deep brain structure), extensive cortical feedback, and in vivo study is complex in delicate early stage animals. Here, we use a novel visual critical period model in larval zebrafish. The advantages to our zebrafish model are that visual plasticity can be demonstrated using a straightforward visuomotor behavior, absence of a cortex and thalamic feedback, larvae are robust and amendable to in vivo study, and the whole brain is optically accessible –providing a unique thalamus-centric vertebrate model for visual plasticity. Last, our prior work has demonstrated that genetically-defined thalamic neurons encode visual experience and display asymmetric patterns of activity that correlate with behavioral performance. Therefore, we have a system where visual experience, thalamic physiology, and changes in the performance of visuomotor performance can be correlated in single animals. We will use this system to determine how visual experience instructs changes to thalamic function through the entire visual critical period until behavioral onset (Aims 1). Changes in inhibitory signaling are well-established to regulate the duration of cortical visual plasticity, yet the role of inhibition in the thalamus is incompletely understood. In Aim 2-3, we will take advantage of our new system to define how visual experience modulates inhibitory input and development in the thalamus. The zebrafish model positions us to address current gaps in knowledge about the basic mechanisms that drive critical period plasticity in the thalamus, and to directly correlate experience with behavioral output. The lab has an established track record of undergraduate training, productivity, and pursuing further STEM opportunities including terminal biomedical degree training or biomedical related careers. This proposal will support continued undergraduate involvement in research, with an emphasis on gaining hands-on practical experiences that create a foundation for long-term success in STEM.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Misdiagnosis is prevalent in younger women with dense breast tissue receiving breast cancer screening, resulting in missed cancers, needless follow-up testing, anxiety, and medical costs. Compared to mammography, magnetic resonance imaging (MRI) detects more breast cancers but still suffers from high false positive rates due to the conventional contrast agents used, e.g., gadolinium (Gd)-chelates. Our long-term goal is to develop novel, safe contrast agents for early detection of breast cancer that reduce the false positives and false negatives of breast MRI. The poor performance of Gd-chelates results from their lack of targeting and constant MRI signal. By remaining active, Gd-chelates produce high background signal in normal tissues and highlight both benign and malignant tumors. To address our long-term goal, we have developed Nano-, Encapsulated Manganese Oxide (NEMO) particles that will provide superior replacements for Gd-chelates. Our preliminary data shows that NEMO particle specificity is achieved by adding peptide targeting to underglycosylated mucin-1, which is overexpressed exclusively on breast cancer cells. NEMO particles provide a unique pH-switchable signal that is only activated upon internalization in acidic tumor cell endosomes (pH 5). No MRI signal is produced at pH of the blood (pH 7.4) or tumor extracellular space (pH 6.5). Our in vivo studies demonstrate that NEMO particles are safely tolerated in mice and exhibit a stronger signal than Gd-chelates. Currently, no high throughput method exists for testing new MRI contrast agents that predicts in vivo performance. The goals of the current project are to develop an innovative portable apparatus to enable evaluation of the sensitivity, specificity, and safety profile of NEMO particles vs. Gd-chelates using MRI and optical imaging of 3D microfluidic tumor models. Our central hypothesis is that NEMO particles will elicit low toxicity, specifically label breast cancer cells, and yield higher MRI contrast compared to Gd-chelates in 3D microfluidic tumor models and in mice with breast cancer. Our hypothesis will be tested with two aims: 1) Evaluate NEMO particle vs. Gd-chelate MRI contrast in 3D microfluidic tumor models and mice. 2) Evaluate toxicity and distribution of NEMO particles in 3D microfluidic tumor models and mice. This project is innovative because NEMO particles uniquely respond to endosomal pH to generate contrast only inside breast cancer cells to provide a simple binary readout (benign “OFF”, malignancy “ON”). Previously developed pH-sensitive MRI contrast agents respond to the acidic extracellular space, which is similar in benign and malignant tumors. We will also create a novel apparatus to enable MRI of 3D microfluidic tumor models for the first time. The proposed research is significant because we will demonstrate that NEMO particles have superior specificity, signal strength, and safety compared to Gd-chelates. This R15 award will offer cutting- edge training to undergraduates in nanomaterials and medical imaging research at West Virginia University. Over 3 years, 6 undergraduates pursuing engineering or biomedical sciences degrees will be recruited, trained, and assessed. This work will lead to further preclinical development and clinical trials of NEMO particles.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT: Increasing evidence suggests that brain perivascular spaces play important roles in intracerebral fluid transport and brain homeostasis. Enlarged perivascular spaces (PVS) have reported associations with aging, mild cognitive impairment (MCI), and Alzheimer’s disease (AD). However, the utility of this biomarker as an imaging predictor of AD is not fully known. To advance progress in AD, early disease biomarkers must be developed. PVS are linked with biological changes that occur early during the course of AD, including dysfunctional intracerebral fluid regulation, neuroinflammation, and small vessel disease. Yet, substantial gaps in knowledge persist with regard to optimal methods of PVS detection and quantitation in live humans. To advance knowledge on PVS effects in humans, a harmonized, non-biased, and time-efficient technique of PVS quantification must be employed in diseased cohorts. In this project, an open-source automated Frangi filter technique of PVS detection will be used to detect PVS and assess their associations with AD in a large, well characterized, underserved Appalachian community cohort. PVS will be analyzed globally and in specific brain regions in aging, MCI, and AD and PVS longitudinal changes will be examined. This work will leverage a rich resource of existing clinical and imaging material available from the Rockefeller Neuroscience Institute Memory Health Clinic and will produce novel data pertaining to PVS metrics. The hypothesis is that PVS serve as a predictor for AD. In Aim 1, we will examine the associations of static PVS metrics with a diagnosis of AD and cognitive decline. In Aim 2, we will examine the associations of longitudinal PVS metrics with a diagnosis of AD and cognitive decline. In both aims, we will investigate how relationships of PVS metrics differ by age, sex, education history, and comorbid diseases. Overall, these studies have the potential to uncover new knowledge regarding optimal methods of PVS measurement, interpretation, and detection in persons at risk of developing AD. This project may reveal new diagnostic and prognostic factors for AD while elucidating appropriate methods for studying PVS in the setting of other age-related neurological conditions.

NIH Research Projects · FY 2025 · 2024-06

Project Summary This project will develop a new tool, SpliceTag, for conditional knock-in/knockout and demonstrate its utility. SpliceTag uses highly cell type specific alternative exons as a vehicle for carrying protein and RNA tags for conditional labeling, or premature stop codons for protein knockout. It can be introduced in an intron of the target gene through CRISPR/Cas9 induced homology directed repair. The SpliceTag exon will then be spliced in mRNA labeling the protein in the desired cell type. In Aim 1 we will create a mouse line in which we use a SpliceTag in Rpl22 to label ribosomes with ALFA peptide in photoreceptor cells. The photoreceptor specific SpliceTag is based on Ttc8 microexon 2a which we have shown in the past to be highly specific to photoreceptor cells. To demonstrate the utility of our approach we will use the Rpl22 SpliceTag line to analyze translational efficiency in photoreceptor cells and how translation is controlled during the diurnal cycle. In Aim 2 we will determine the feasibility of using SpliceTag to label proteins in epithelial cell and inner retina neurons. We will also determine if it can incorporate commonly used tags, SNAP and CBP/Strep, that are significantly larger than the Ttc8 exon 2a on which SpliceTag is based. Since SpliceTag does not rely on recombinases it can enable experimental designs where recombinase and floxed alleles are used for genetic manipulations in parallel with SpliceTag, including knockouts in cell types other than the cell type in which the protein is being labeled.

NIH Research Projects · FY 2025 · 2024-04

ABSTRACT The United States deployed ~3 million service members to the Middle East since 2001. Approximately 600,000 Veterans now suffer from “Chronic Multisymptom Illness” (CMI). The “Promise to Address Comprehensive Toxics or PACT Act, signed into law in 2022, now exists to mitigate Veteran suffering associated with military burn pit exposures. The single most common risk factor among these ailing Veterans is inhalation exposure to complex combustion emissions generated by these burn pits. CMI symptoms include: cardiopulmonary morbidity, cognitive impairments, behavioral disorders, fatigue/diminished energetics, compromised immune function and pain. Despite the fact that the number of Veterans suffering from CMI is forecast to surge, two major knowledge gaps exist: 1) the exposure conditions that lead to CMI are poorly characterized, and 2) the mechanism(s) of CMI development cannot currently be studied as a model of exposure does not exist. Neither of these gaps can be properly addressed in the absence of an emission generator capable of mimicking burn pits and their diverse parameters of operation. The objective of this application is to validate our novel generation system and identify the most relevant fuel mixtures that accurately recapitulate military burn pit emissions. This will be achieved in our unique Inhalation Facility located in the WVU Center for Inhalation Toxicology. AIM 1: Determine, optimize and validate the burn pit surrogate emission generator parameters of operation. We have developed an automated combustion chamber with a hopper feed system to operate under a variety of temperatures, feed speeds, and air richness. Combustion is enhanced by a fuel feed system that independently drips jet fuel (the most common accelerant used in burn pits) into the combustion chamber. The goal is to identify the full range of these parameters, and couple them with the resultant emissions delivered to the exposure chamber for real-time aerosol characterization and sampling. AIM 2: Determine the operable proportions of representative mixed fuels that when combusted, produce reliable and repeatable emissions for real-time aerosol characterization and inhalation exposures. We manufacture combustible pellets with a variety of raw materials to feed into our surrogate emission generator. These materials have different combustion temperatures, and varying the mixture and/or amount/pellet produces different emissions. The goal is to identify a range of mixture proportions that combust and smolder over the full operating parameters, that produce reliable and repeatable emissions in the exposure chamber. Upon completion, a novel inhalation exposure instrument will be validated and optimized for subsequent CMI studies. Fuel mixtures and accelerant delivery rates will be established as the standards for these studies. Identification of these parameters is critical for rigor and reproducibility, and will also initiate the foundation for future CMI research that ultimately benefits Veteran health. Added value exists as the surrogate generator is capable of combusting virtually any substance. Therefore, it will also be invaluable in assessing first- responder exposures to diverse conditions such as domestic and wildland urban interface (WUI) fires.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Breast cancer is a devastating disease that affects large numbers of women annually; it is estimated that 1 in 8 women in the United States will be diagnosed with this disease during their lifetime. The five-year survival rate for women diagnosed with localized or regional breast cancer is greater than 90 percent. However, the presence of brain metastases reduces five-year survival rates to less than 10 percent. Chemotherapy treatment of brain metastases is challenging and has yielded inferior results compared to tumors in the periphery, likely reflecting the inability of chemotherapy to cross the blood brain barrier (BBB) and/or blood tumor barrier (BTB) at efficacious rates. Recent studies demonstrate circadian regulation of BBB permeability; however no study has examined whether temporal alterations in chemotherapy administration can improve the efficacy of treatment of brain metastases. This project aims to take advantage of circadian control of BBB permeability by optimally timing chemotherapy administration to increase anti-tumor efficacy and reduce adverse side effects of brain metastases. Specifically, I hypothesize that circadian control of efflux transporter expression at the BBB underlies the fluctuations in BBB/BTB permeability to chemotherapeutic agents. Permeability of two of the most commonly prescribed chemotherapeutic drugs for breast cancer, doxorubicin (A) and paclitaxel (P), will be assessed via phosphor autoradiography imaging by using 14C labeled chemotherapeutic drugs. Additionally, pharmacological inhibition and genetic approaches (CRISPR) will be utilized to determine both the type and location (BBB or BTB) of efflux transporters that underlie altered permeability. Mice harboring brain metastases of breast cancer will receive intravenous injections of doxorubicin/cyclophosphamide cocktail or paclitaxel every 2 weeks during either the peak or trough of BBB permeability. Anti-tumor efficacy will be assessed via bioluminescence and immunohistochemistry. Adverse behavioral effects of chemotherapy will be assessed via multiple behavioral tasks and sleep assessment. I predict that optimal timing of chemotherapy administration will increase anti-tumor efficacy and minimizes adverse side effects. Indeed, preliminary data demonstrate that altering only the timing of injection increased the amount of chemotherapy within brain metastases of breast cancer by approximately 50%. Thus, chrono-chemotherapy represents a viable and novel treatment strategy. Together, these studies will provide essential information with a high potential for clinical relevance to better treat patients with breast cancer. This career development training award will enable me to become an independent investigator by allowing training in emerging techniques (i.e., CRISPR and blood-brain barrier/blood-tumor barrier biology), professional development, grant writing, guest lab training, mentor training, and highly focused research support. To help accomplish my goals, I have assembled a multidisciplinary mentoring team that has extensive expertise in the fields of circadian biology, cancer, and neuroscience.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Multiple sclerosis (MS) is an immune-mediated disease that impacts approximately 2.3 million people world- wide. MS is caused by the activation and the complex interactions between different immune cell types that cause inflammation in the central nervous system (CNS), which leads to demyelination and axonal degeneration. A validated animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), has been utilized to demonstrate that the cognate interactions between CD4+ T cells and monocyte-derived cells (MDCs), defined here a mixed population of dendritic cells and macrophages differentiated from monocytes during the autoimmune-driven neuroinflammation, are required for the pathogenesis of EAE. The overall goal of this proposal is to identify signaling pathways and their downstream effector mediators that regulate the interactions of CD4+ T cells and MDCs within CNS that promote the pathogenesis of EAE. Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) play a critical role in mediating cellular responses following stimulation of cytokines, interferons, growth factors. The activated STAT5 proteins can form dimers and tetramers. The biological functions of STAT5 tetramers are not fully understood. Using a Stat5a-Stat5b N-domain double knock-in (DKI) mouse strain, in which STAT5 tetramers cannot be formed but STAT5 dimers are unaffected, we found that STAT5 tetramers promote the pathogenesis of EAE. The mild EAE phenotype in DKI mice correlates with reduced interactions of CD4+ T cells and monocytes/MDCs in the spinal cord meninges during EAE. We further demonstrated that STAT5 tetramer activation by cytokine GM-CSF promotes monocyte differentiation into dendritic cells and the expression of chemokine CCL17. Correspondingly, the expression of CCL17 in the spinal cords is significantly reduced in DKI mice during EAE. Importantly, mice receiving STAT5 tetramer-deficient Th17 cells treated with CCL17 developed more severe EAE with earlier onset of the disease compared with mice receiving cells without CCL17 treatment. Our central hypothesis is that GM-CSF-mediated STAT5 tetramerization is critical for facilitating the interactions of pathogenic Th17 cells and monocytes/MDCs in the spinal cord meninges via a CCL17-dependent mechanism, and these cell interactions are required to promote the pathogenesis of EAE. We will test our hypothesis in three specific aims. In Aim 1, we will utilize different EAE induction strategies to determine the pathogenic role of STAT5 tetramers in Th17 cells and monocytes/MDCs during EAE. In Aim 2, we will test our hypothesis that STAT5 tetramer signaling promotes monocyte differentiation and Th17 cell-MDC interactions at the spinal cord meninges during EAE using intravital imaging technique. In Aim 3, we will investigate the mechanism by which CCL17 promotes the pathogenesis of EAE. Specifically, we will test our hypothesis that CCL17 increases the affinity of integrin VLA-4 on Th17 cells, thereby facilitates Th17 cell-MDC interactions. Targeting the pathways and mediators that control the interactions between CD4+ T cells and MDCs within CNS may become a novel strategy for the treatments of MS.

NIH Research Projects · FY 2025 · 2023-09

For 28 years the Health Sciences and Technology Academy (HSTA) of the West Virginia University (WVU) Robert C. Byrd Health Sciences Center with the help of the National Institute of Health (NIH) Science Education Partnership Award (SEPA), has implemented a one-of-a-kind mentoring program designed to assist WV high-school students to enter and succeed in Science, Technology, Engineering, and Mathematics and Medicine (STEM+M) based undergraduate and graduate degree programs. HSTA submits this proposal with the novel approach of using a team-based mentoring structure to facilitate the development and execution of Community Based Participatory Research (CBPR) and Citizen Science (CI) projects, to be carried out by HSTA students. HSTA marshals and links the efforts of an impressive network of mentors across educational levels to support 160 junior students annually (800 over a five-year period) through HSTA TEAMS. The innovation of HSTA TEAMS is in the combined use of the flexible structure of HSTA and the long arc of mentorship linkages, connecting college to community through high school, and now middle school students, in over half of WV counties across the state. It is the flexible structure of HSTA that allows the HSTA BioMed Summer Institute and HSTA community-based after-school club curriculum to respond to what is happening on the ground for HSTA families and communities, as well as with what the research community is observing and investigating. The Biomedical Summer Institute at WVU for teachers, mentors, and students directed by HSTA leadership and staff in collaboration with university faculty sets the foundation for the HSTA academic year community-based after school clubs during which specially trained HSTA teachers mentor students into research. Through the after-school club curriculum HSTA students will use validated resources, supported by HSTA TEAMS of mentors, to design STEM+M educational interventions or experiments with middle school students. The results of the STEM+M community-based research projects will be disseminated through regional and state HSTA research symposia, as well as through national conferences and publications.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Accumulating evidence suggests that following ischemic stroke hypoperfused brain tissue is functionally disabled as electrical communication among penumbral neurons is disrupted due to marked reductions in oxidative metabolism. Thus, it is apparent that mitochondrial dysfunction plays a central role in the degree of neuronal cell death encountered following ischemic brain injury; however, mitochondria have been slow to be fully investigated. Through a serendipitous discovery of an off-target therapeutic effect of pioglitazone, a small mitochondrial iron-sulfur cluster protein, mitoNEET (mNT) was identified that has renewed interest in therapeutic targeting of mitochondria. MitoNEET is embedded in the outer mitochondrial membrane and acts as a redox and pH sensor to regulate mitochondrial bioenergetics, especially in response to cellular stress. Using pioglitazone as our parent compound, we designed NL-1, a first-in-class ligand with high specificity for mN. Using NL-1, we have demonstrated marked improvements in stroke neuropathology and functional impairment following transient middle cerebral artery occlusion (MCAO) in mice and rats. The objective of this proposal is to address fundamental gaps in knowledge regarding how mNT works within the brain to mitigate acute ischemic brain injury. Our central hypothesis is that modulation of mNT acts to improve vulnerable neurons within the penumbra by reducing the vicious cycle of excessive iron-induced lipid peroxidation and increased neuronal death. Based on a strong body of prior literature and pilot data, we postulate the initial target of activity for NL-1 is the cerebral microvasculature; thus, we propose two specific aims to test our hypothesis. In specific aim 1, we will test if mNT selective ligand, NL-1, mitigates ferroptosis using a 4 cell Transwell in vitro model of the blood- brain barrier following oxygen-glucose deprivation with reperfusion. Whereas, in specific aim 2, we will test if mNT ligand, NL-1, reduces brain iron accumulation and blood-brain barrier dysfunction post-MCAO. Successful completion of the proposed research is expected to provide a: (1) greater understanding of how & where mNT mitigates brain injury following ischemic stroke; (2) new insight into the impact of mitochondrial dysfunction & diminished bioenergetics on ischemic stroke outcomes; & (3) strong scientific foundation for an interventional therapeutic approach for treating ischemic stroke. The mechanistic & preclinical data obtained through these studies will serve as critical milestones for advancing the development of mitochondria-targeted therapies for treating neurological injuries & disease.

NIH Research Projects · FY 2025 · 2023-08

Robert Stansbury, M.D., is a fourth generation West Virginian and pulmonologist whose overarching career goal is to improve healthcare disparities for chronic respiratory illness in rural communities. This K23 mentored career development award, entitled “A Novel OSA Training Intervention to Improve Provider Management of Sleep Apnea in Rural Communities”, builds upon my previous training to ensure a successful transition into being an independent investigator. My research career goals include: 1) obtain training in mixed-methods research to investigate facilitators and barriers to care for respiratory disease in rural areas, 2) receive training in implementation science using community engaged research and ultimately conduct clinical trials to improve respiratory disease outcomes, and 3) enhance management skills for developing and leading an independent research program. I am a non-traditional candidate and have made significant contributions as a faculty member at West Virginia University (WVU). My recent administrative work as interim Chief for the Division of Pulmonary, Critical Care and Sleep Medicine at WVU has led to a pivot from research in respiratory physiology to healthcare disparities. I have completed recent studies in this area; however, this research has been completed with extensive mentorship and support. This career development award is an ideal mechanism for me to successfully transition to lead my own independent research program in respiratory health disparities. With my primary mentor, Dr. Pat Strollo, I have assembled a strong team of co-mentors and consultants who will guide me through the proposed career development and research training plans. Clinical, didactic, and research collaboration with these mentors and consultants forms the foundation of these career development activities and will facilitate a multidisciplinary team approach. This team has expertise in obstructive sleep apnea, education, academic career development, clinical and translational science and rural health/healthcare disparities. The career development training plan utilizes intellectual resources and the research infrastructure available at WVU, West Virginia Clinical and Translational Sciences Institute (WVCTSI), and the University of Pittsburgh Medical Center (UPMC) where I have an adjunct faculty appointment. I will attend national conferences and pursue further training outside of WVU when optimal training is not available locally. The WVU is an ideal location to study health care disparities in rural communities due to the long-standing challenges of this rural state. The university has a world-class research infrastructure, an extensive network of hospitals and clinics, and a large tertiary care university hospital which serves a predominant rural population. This K23 award will enable my successful transition to being an independent physician-investigator with a research program addressing the healthcare disparities of respiratory illnesses found in rural communities.

NIH Research Projects · FY 2025 · 2023-08

There are currently nearly 8,000 clinical trials and observational cohort studies funded by the National Institutes of Health in the United States, yet less than 10% of these studies are conducted in Institutional Development Award (IDeA) states that serve underrepresented minority and rural populations. Not surprisingly, only approximately 7% of expenditures for clinical trials and observational studies go to the 23 IDeA states and Puerto Rico. Barriers that limit expansion of clinical trial and cohort studies in IDeA states include lack of effective communication to trial sponsors of the expertise and capability that IDeA institutions possess to effectively conduct clinical studies, effective communication to IDeA investigators regarding clinical trials opportunities, and lack of clinical trial coordinators. The overall goal of this project is to increase numbers of clinical trials and observational cohort studies in IDeA states, thereby increasing trial availability and participation of minority and rural populations historically underrepresented in clinical trials. We will achieve this goal by establishing the IDeA State Consortium for Clinical Research Resource Center (ISCORE-RC) comprised of the Clinical Trials Service Center and the Clinical Research Coordinator (CRC) Development Program to address the identified barriers through accomplishment of the following specific aims. Aim 1 - Effectively communicate and market to clinical trial sponsors the expertise, quality, and capacity of IDeA state institutions to conduct clinical trials. We will accomplish this aim through several tactics to include leveraging the TriNetX platform that connects clinical trial sponsors with institutions serving relevant patient populations to identify sites with potentially eligible participants for specific clinical trials, creating a repository containing site-specific profiles showcasing strengths of individual IDeA state trial groups, establishing an ISCORE-RC website that provides a public resource to demonstrate benefits of partnering with IDeA organizations to increase clinical trial participant representation from underserved populations, and facilitating networking of sponsors with investigators. Aim 2 - Communicate to IDeA state investigators clinical trial opportunities. To accomplish this goal, we will develop site specific search criteria in clinicaltrials.gov that will allow the ISCORE-RC Clinical Trials Service Core to monitor relevant clinical trials of interest currently in the participant recruitment phase. Aim 3 - Train a cadre of skilled clinical research coordinators. Entry into this program will be open to experienced clinical personnel as well as to non-clinical individuals. Both part-time and full-time effort of trainees will be accommodated. The program will include an online didactic portion, journal club, and seminar series as well as a prominent experiential component to be conducted at participating sites. Upon completion of the core content, participants will receive a CRC basic training certificate and will be eligible for further training towards an advanced training certificate and membership in the ISCORE-RC CRC Academy, a networking and professional development platform that includes current research coordinators at participating sites as well as trainees.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY The distribution of engineered nanomaterials (ENM) in consumer products, manufacturing processes and clinical diagnostics is rising rapidly, despite our limited understanding of their impacts on human health. ENM exposure is of particular concern during fetal development and it can influence susceptibility to pathological insults later in life. Mitochondria play an important role in fetal developmental and they can be impacted by environmental conditions, which has led to the novel concept of mitochondrial programming. Epigenetic changes are important determinants of mitochondrial programming, as they influence the organelle's proteomic make-up, which is responsible for its structure, function and redox balance. Nevertheless, mitochondrial programming in the context of development is understudied. Our laboratory made the initial observation that maternal ENM inhalation exposure causes cardiac contractile dysfunction and disruption to mitochondrial bioenergetics in the developing fetus. These effects were sustained into adulthood. We also reported that maternal ENM inhalation exposure increases epigenetic methylation of mRNAs in the fetal heart. MRNA methylation occurs primarily to adenosine leading to N6-methyladenosine (m6A), and to a lesser extent to cytosine, leading to 5-methylcytosine (m5C). The preliminary data in this grant application suggest that maternal ENM inhalation exposure influences fetal cardiac mitochondrial programming by enhancing oxidant production and mitochondrial dysfunction, but it is unclear whether this is mechanistically linked by epigenetic methylation to nuclear genome-encoded mitochondrial mRNAs and loss of mitochondrial proteins. The proposed studies focus on this gap in knowledge and they are designed to determine whether maternal ENM inhalation exposure negatively influences mitochondrial programming in the fetal heart and the susceptibility to future cardiac pathological insult, through an oxidant driven mechanism. The studies address this specific need, as they will identify mechanisms driving fetal mitochondrial dysfunction resulting from maternal ENM inhalation exposure as well as the susceptibility to a secondary cardiac pathological insult that occurs later in life. The central hypothesis being tested is that maternal ENM inhalation exposure epigenetically reprograms fetal cardiac mitochondria through an oxidant- driven mechanism that results in enhanced susceptibility to a secondary cardiovascular insult at adulthood. The objectives of this application are to determine the influence of maternal ENM inhalation exposure and the impact of enhanced oxidant scavenging on (1) fetal cardiac mitochondrial programming that influence mitochondrial structure, function and redox balance; (2) fetal cardiac epigenetic methylation of nuclear genome-encoded mRNAs that encode for mitochondrial proteins; and (3) the susceptibility to a secondary cardiovascular insult at adulthood. Completion of these studies is expected to provide fundamental mechanistic insight regarding fetal mitochondrial programming in maternal exposure models and the susceptibility to future cardiac pathologies.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Late-stage, HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC) (or HNC-) is the most lethal form of HNSCC. HNC- occurs at the highest incidence in the Appalachian region, where a large segment of this population is rural, economically disadvantaged and medically underserved. The Appalachian population is the highest user of tobacco in the nation, a behavior directly linked to disparities in HNC- incidence and mortality. As such, there is a pressing need to identify the underlying genomic mechanisms responsible for the disparate survival in tobacco-positive Appalachian HNC- patients as an essential step towards improving medical outcomes for these patients. Single gene informatic analysis of national cohorts has identified elevated copies of multiple genes associated with smoking. The majority of these genes map to chromosome 11q13 cytobands, the most commonly amplified region in HNC- and long known to be associated with reduced survival. 13 smoking-correlated genes overexpressed from the 11q13 amplicon correspond with decreased survival, increased risk of death and elevated risk of lymph node metastasis. While some genes in this region have been well studied as drivers of HNC- progression, other genes identified by this analysis have unknown roles in cancer and may present new targets for therapeutic intervention. The overall goal of this proposal is to identify the roles of uncharacterized genes from the smoking-associated expression signature (SAES) in the 11q13 amplicon that contribute to neoplastic progression of Appalachian tobacco-positive HNC-. Our central hypothesis is that overexpression of novel 11q13 genes in the SAES contribute in driving reduced Appalachian HPV-survival. Oncogenic screening of uncharacterized SAES genes will be conducted to evaluate the individual contributions of each gene in promoting cancer hallmarks. SAES gene function will be assessed using cultured cells from Appalachian tobacco-positive patients and mouse orthotopic HNSCC models. Aim 1 will test the role of SAES genes in driving tumor cell growth, proliferation, invasion, and metabolic reprogramming in tobacco-induced 11q13 amplified HNC-. Aim 2 will utilize experimental and clinically-parallel imaging modalities to individually evaluate novel predicted drivers of lymph node metastasis. These genes will be tested for promoting tumor cell growth, nodal spread, altered metabolism and proliferation in 11q13 amplified Appalachian HNC- tumors. A comprehensive understanding of how each novel SAES gene contributes to enhancing this aggressive HPV- subtype will fill a key gap in our knowledge regarding how the 11q13 amplicon contributes to the overall poor outcomes seen in tobacco-associated HNC-. Results from this proposal will provide a foundation for future studies that will mechanistically address novel tumor-promoting SAES genes identified from this work as drivers of disease aggressiveness, potentially serving as new targets for therapeutic development and/or biomarkers to improve treatment of this highly refractory disease in the Appalachian population.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT At risk youth (ARY) are those who are at risk for suicide and self-harm. Suicide is the second highest cause of death among adolescents, but parent support and communication can improve mental health outcomes for this population. There are few formal support programs to help parents navigate conversations with their children about suicide and self-harm. This K23 project will: 1) survey a cross-section of parents of ARY in the United States to better understand their experiences, 2) design a stakeholder-informed online support intervention including peer support, and 3) pilot test the online support intervention for feasibility and acceptability. Conduct of these aims, in addition to a career development plan inclusive of coursework, workshops, and leadership development, will allow for the applicant’s academic growth in nationally representative surveys, stakeholder-engaged intervention development, clinical trials, and research program leadership, and will further advance the applicant’s transition to independence. These aims will also provide the necessary data to conduct a future R01 randomized control trial to evaluate the effectiveness of the stakeholder-designed intervention to improve parent confidence in supporting ARY. TITLE Development and Feasibility Trial of Online Peer Support for Parents of At Risk Youth MAJOR GOALS OF THE PROJECT Aim 1: Identify factors associated with supporting youth in a national sample of parents. Identifying parent factors associated with supporting youth is critical to design parent support interventions that are feasible and effective at increasing parent support and improving youth health. I will conduct an online, national, cross-sectional study of parents of ARY to identify factors associated with parent support. Aim 2: Develop a stakeholder-informed online support intervention for parents of ARY. Stakeholder input is vital for creating an acceptable support intervention to help parents support their children and improve health outcomes for ARY. Using a community engaged intervention mapping approach, I will continue to conduct semi-structured individual interviews of up to 40 parents to understand key stakeholder perspectives on the core features and design of an online parent support intervention. An iterative user-centered design process will be used to develop the intervention currently planned to consist of a 1:1 peer network. Aim 3: Assess feasibility and acceptability of the intervention with parents of ARY. Parents of ARY seek support and resources online and assessing feasibility and acceptability of an online support intervention for these parents is needed before further exploration of intervention effects. I will pilot the stakeholder-informed intervention in a randomized feasibility trial with parent-ARY youth dyads to assess their perspectives regarding its feasibility and acceptability compared to control dyads accessing an existing online information page for families of ARY. I will use the RE-AIM Framework to assess usability and acceptability of the intervention and website through qualitative and quantitative assessments. CURRENT MENTORING TEAM QUALIFICATIONS Primary Mentor Dr. Alfgeir Kristjansson has extensive experience in adolescent suicide risk assessment and prevention through his youth behavioral development work both in the United States through the West Virginia Prevention Research Center and in Iceland through his development of the Icelandic Prevention Model. Co-Mentors Dr. Nadia Dowshen and Elizabeth Miller have expertise in adolescent development and mental health as both adolescent medicine specialists and researchers. Dr. Dowshen’s research has included suicidality assessments and the development of support interventions specific to adolescents and young adults. Dr. Miller’s research has included mental health assessments and intervention development including supporting youth with suicidal ideation. This includes involvement in the World Mental Health Consortium. NEW CONSULTANT Dr. Stephen Deci is a board-certified child and adolescent psychiatrist and the Interim Division Chief of the Child & Adolescent Division in the Department of Behavioral Medicine at the West Virginia University School of Medicine. Dr. Deci’s clinical expertise is in adolescent suicide prevention and he is the Medical Director of the Adolescent Inpatient Mental Health Unit in the Chestnut Ridge Psychiatric Hospital where he cares for adolescents who considered or who have attempted suicide. He has extensive connections to patients and families impacted by suicide and self-harm and will ensure I am well connected to these communities.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Cardiovascular complications account for the majority of deaths in diabetic patients. Mitochondrial dysfunction is central to the disease and it precipitates contractile impairment, leading to death. However, the precise mechanisms that cause mitochondrial dysfunction in the diabetic heart remain unclear. Using type 2 diabetic human (patient) and mouse (db/db) models, we observed pronounced disruption to mitochondrial structure and function, which were associated with the loss of mitochondrial proteins. The vast majority of mitochondrial proteins are nuclear genome-encoded and require import into the mitochondrion. Import occurs through a coordinated set of machinery containing an active motor, driven by mitochondrial heat shock protein 70 (mtHsp70). We observed decreased mtHsp70 content in cardiac mitochondria from type 2 diabetic patients and db/db mice, and a decrease in mitochondrial protein import. Following import, mitochondrial proteins are refolded into native structures to become functional. MtHsp70 also participates in the refolding process, and works synergistically with Lon Peptidase 1, Mitochondrial (LonP1), an AAA+ protease of the mitochondrial matrix. We have also observed a decrease in LonP1 in the type 2 diabetic heart. When mitochondrial protein import is not functioning properly, aggregated nuclear genome-encoded proteins accumulate on the exterior of the mitochondrion leading to a phenomenon termed mitochondrial precursor over-accumulation stress. Currently, it is unclear what factors contribute to a decrease in protein import efficiency and refolding or whether manipulation of these processes can restore mitochondrial proteomic make-up, mitochondrial function and cardiac contractile performance in the diabetic heart. Our proposed studies address this critical gap in knowledge. The information will enhance our understanding of these processes and aid in the development of therapeutic strategies that target specific import constituents that contribute to loss of mitochondrial proteins. The central hypothesis to be tested is that decreased protein import and refolding in the type 2 diabetic heart causes loss of mitochondrial proteins and mitochondrial precursor over-accumulation stress leading to mitochondrial dysfunction and contractile impairment. The objectives of this application are to (1) identify submitochondrial locations where protein import is compromised in type 2 diabetic mitochondria and the impact on import machinery; (2) evaluate the impact of type 2 diabetes mellitus on mitochondrial protein refolding and the synergistic influence of mtHsp70 and LonP1; and (3) determine the extent of mitochondrial and proteomic stress that occurs in the type 2 diabetic heart, due to failed mitochondrial protein import contributing to mitochondrial precursor over-accumulation stress. Completion of these studies is expected to provide fundamental molecular insight into the mechanisms contributing to the loss of nuclear genome-encoded mitochondrial proteins in the type 2 diabetic heart and the cellular consequences leading to mitochondrial and cardiac dysfunction.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY (See instructions): The incidence of tick-borne diseases (TbD) is increasing, with ~500k reported cases during 2004-2016 from the National Notifiable Diseases Surveillance System of the Centers for Disease Control (CDC), and is expected to increase due to global climate change. Some of the most common TbD in the U.S. include anaplasmosis, babesiosis, and Lyme disease transmitted by the same tick vector, I. scapularis. Lyme disease is especially pernicious because it is difficult to diagnose early, often misdiagnosed, and is difficult to treat. Because current diagnostic methods are insufficiently sensitive or non-existent, we propose a novel approach for diagnosis via a microfluidic platform with an integrated multimodal sensing system and machine learning (ML) algorithm. Based on our preliminary and published data, we hypothesize that we can detect TbD and their coinfections from whole blood. Central to our vision is a system designed for minimal user intervention to detect and measure complex cell data using ML. The diagnosis of TbD will be achieved through three objectives: 1) design a dielectrophoresis (DEP) based platform for detecting TbDs from whole blood; 2) design 3D sensors and a readout integrated circuit (ROIG) for sensitive in-vitro detection of cells; and 3) develop an ML algorithm to diagnose early-stage Lyme disease, babesiosis, and anaplasmosis. An extremely sensitive (<0.1aF), low-voltage (1.8V for core & 3.3V for 10), low-power (<10mW), multi-channel (~16-ch), high-speed (~5MSample/sec per channel) readout integrated circuit (ROIG) will be integrated to detect single-cell behavior at high resolution (~10-bit resolution). The novel ML algorithm(s) will use cell data to determine the type of infection intelligently, reducing >96Gbit data during each diagnostic cycle (~2min) into as simple as 1-byte diagnostic information (i.e., 1: positive, 0: negative) per tested population of the cells or disease. Further, an open-source program to diagnose TbD will be created and made available freely from a public software repository. This developed diagnostic technology will simultaneously meet high-sensitivity, low-power/voltage, high-purity, and high-viability performance metrics by cutting the diagnosis time from 4-6 weeks (late stages) to <30 min (early stages). The proposed research will lead to faster diagnosis, reducing the hospitalization, morbidity, and mortality associated with TbD and their coinfections.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Elizabeth Bowdridge, Ph.D., is a reproductive toxicologist whose overarching career goal is to establish a successful laboratory focused on the effects of occupational and environmental factors on microvascular adaptations during gestation that ultimately impact fetal outcomes. Her proposed project combines an occupationally relevant inhalation exposure model during gestation and epigenetic analysis of offspring, which will establish the impact of hormones and vascular adaptations that are critical for healthy reproductive outcomes. Acquiring this knowledge is a critical step for ensuring optimal maternal and fetal health. Career Development Plan: Dr. Bowdridge is a Research Assistant Professor in the Department of Physiology and Pharmacology at West Virginia University (WVU). The proposed career development plan will build upon her previous training with four training goals to enhance her trajectory toward becoming an independent investigator: 1) Acquire advanced training in placental pathologies and identification of nanomaterials using techniques such as FESEM and TEM; 2) Broaden my knowledge of how the dose of specific toxicants are applicable in an occupational setting and ways in which these affect critical windows of embryonic and fetal developmental, oxidant formation, and epigenetics; 3) Increase technical expertise in occupational exposure relevance, dose deposition and epigenetic analysis across generations; 4) Develop professional skills in grant writing, research and laboratory leadership, statistical analysis, and research ethics. Mentors/environment: Dr. Bowdridge and her primary mentor, Dr. Timothy Nurkiewicz, Ph.D., have assembled a strong team of co-mentors and advisors to guide her through the proposed training and research activities. The proposed career development plan utilizes the intellectual, microscopy, free radical biology, and genomics resources available through WVU and at the National Institute for Occupational Safety and Health. The WVU Genomics and Bioinformatics cores provide centralized genomic and biostatistical analysis training to investigators at WVU and other universities across WV. As an institution WVU is committed to supporting junior faculty members through internal grants, administrative support and structured opportunities for faculty networking and education. Research: Adverse reproductive outcomes, such as miscarriages, are common in pregnant women working in occupational settings. These women are exposed to ENM such as, nano-titanium dioxide (nano-TIO2) via inhalation. One likely, but uninvestigated, way that ENM may mediate these poor outcomes in an occupational setting is by decreasing hormones critical pregnancy hormones such as estradiol (E2). This proposal is the first step in linking E2 and the peptide Kiss with vascular dysfunction and adverse reproductive outcomes due to occupationally relevant maternal nano-TiO2 inhalation exposure. Aim 1 identifies the roles of E2 and Kiss and oxidant load across timepoints in gestation on uterine microvascular function and fetal health following maternal nano-TiO2 inhalation exposure. Aim 2 determines the impact of maternal nano-TiO2 inhalation exposure on reproductive health of F1 female progeny. The completion of these aims will identify the roles of E2 and Kiss in establishing a healthy fetal environment by regulating vascular and endocrine function during occupational exposures.

NIH Research Projects · FY 2025 · 2022-07

Inhalation exposures to a large variety of airborne toxicants are an ever-present, rising and ubiquitous public health threat in our environments whether it be outdoor air pollution, industrial processes, or indoor domestic or occupational toxicants. Inhalation exposures are widely associated with adverse health outcomes in major physiological systems such as: pulmonary (asbestosis, black lung, silicosis); cardiovascular (ischemia, infarction); neural (stroke, behavior), immune (inflammation); and endocrine/reproductive (disruption, developmental origins of health and disease). The whole of West Virginia is part of Appalachia, wherein significant health disparities such as cardiovascular/cerebrovascular disease, obesity, diabetes, metabolic syndrome, depression and addiction are disproportionately high. Further, poor socioeconomic status, and geography (thermal inversions) trap communities in the immediate proximity of mountain-top mining (surface mining) and hydraulic fracturing (“fracking”) operations that produce tremendous amounts of industrial air pollution. An immediate demand exists to train the next generation of scientists able to accurately assess the complex interplay among these toxicological risks, and ultimately improve public health in West Virginia and the nation. The Predoctoral Training in Systems Toxicology Program will formalize and standardize our already strong training programs in the biomedical sciences and focus on inhalation toxicology research. Several innovative aspects of this training program are semester-long didactic courses in “Toxicology” and the “Inhalation & Aerosol Sciences”, a unique “Environmental Immersion” in community outreach/engagement via air sampling downwind of mountaintop/surface mines and fracking platforms, the “Paracelsus Society” colloquium and journal club, an Associate Scholars Program and leadership training. This rigorous training program will select the best doctoral students from the participating Biomedical Sciences Ph.D. and Clinical & Translational Sciences Ph.D. training programs at the West Virginia University (WVU) Health Sciences Center (WVU HSC) and Engineering and will prepare them with the skills, knowledge and acumen needed for a successful career in the various field of toxicology. It is projected that up to 40 trainees will be enrolled in this program during the funding period. The specific training for each mentee will be tailored based on their annually updated Individualized Development Plan (IDP), and a “Career Options” Program will help prepare them for their chosen career in the toxicology field. Program training is expected to last 2-to-3 years. This pre-doctoral training program will create a new generation of young scholars who can directly address the need for innovative toxicology research for the citizens of West Virginia, Appalachia, and the nation.