University Of Nebraska Medical Center

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY The interaction between host and pathogen is antagonistic, and this is best illustrated by infection of host cells by the obligate intracellular bacterium Chlamydia trachomatis (Ctr). Ctr is absolutely dependent on the host cell for numerous indispensable nutrients, a characteristic that manifest its obligately intracellular nature. Metabolic dependence on the host renders Ctr vulnerable to changes in the overall physiology of the host, including fluctuations in metabolic capacity and generation of metabolites essential to Ctr. The host-mediated process of nutritional immunity, which is based on withdrawal of metabolites from the pathogen through cessation of biosynthesis and/or acquisition, or active sequestration, is quite effective in eliminating various types of pathogens, e.g. viral, bacterial, and parasitic. The best characterized foci of nutritional immunity are essential biometals (i.e. iron, manganese, magnesium, etc.), carbon sources (e.g. glycolytic intermediates and products), and amino acids (e.g. tryptophan). Here, we propose that host nucleotide biosynthesis, specifically that of GTP is a novel node of nutritional immunity to negatively impact chlamydial growth. This hypothesis is based on the premise that Ctr is a GTP auxotroph, in that it is unable to synthesize this nucleotide from products of glycolysis and the tricharboxylic acid cycle, and instead acquires guanine nucleotide directly from the host. Several preliminary evidence also illustrate chlamydial modulation of host GTP biosynthesis through modification of the filamentation dynamics of the rate-limiting enzyme IMPDH2. This enzyme is involved in the first step committed to GTP synthesis within the purine biosynthetic pathway, and in Ctr-infected cells, it is regulated at the post- translational level. IMPDH2 enzymatic activity is thought to be significantly enhanced after polymerization of IMPDH2 octamers into filamentous structures. This is a natural process that is associated with increased GTP demands; and examples include rapid cell proliferation, viral infection, and pharmacological inhibition of the purine biosynthetic pathway. Ctr infection also induces filamentation, which is the first demonstrated evidence of this process being induced during bacterial infection. Of particular interest was the different properties of IMPDH2 filaments in Ctr-infected cells, and they include altered filament morphology (rod-like vs. wisps observed in uninfected cells), increased numbers associated with infection, unipolar vs. bipolar filamentation, the former being predominantly observed in infected cells. In addition, preliminary studies revealed that IMPDH2 filamentation dynamics are affected by exposure to interferon-gamma (Ifng), in that the number of filaments induced by inhibition of GTP biosynthesis by mycophenolic acid (MPA) is decreased in Ifng-treated cells. The opposing effects of pathogen and host-derived cytokines on IMPDH2 filamentation dynamics places IMPDH2 in the middle of the battle between Chlamydia and host cell. In Aim 1, the molecular mechanism of chlamydial modulation of IMPDH2 filamentation will be characterized; and in Aim 2, the exciting possibility of IMPDH2 modulation is part of the host interferon-based nutritional immunity will be investigated.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Cancers originating in the peritoneal cavity are especially challenging to treat for a multitude of reasons, including diagnosis at a late stage of disease, relatively few intraoperative methods to assess the extent of disease, and lack of methods to detect tumor margins in real time for patients undergoing curative surgery. Patients with colorectal cancer, a leading cause of cancer-related death, would significantly benefit from knowing better the extent of disease to optimize treatment strategy and to maximize tumor removal during cytoreductive surgery. Recently, the development of fluorescence image-guided surgical systems and optical probes to better identify malignancies has enabled the possibility to intraoperatively guide tumor removal and assess margin status. There are now targeted optical probes for fluorescence-guided surgery. However, these are not designed for CRC. Accordingly, we propose to develop a near infrared fluorophore (NIRF) antibody conjugate that is specific for CRC. Claudin-2 (Cldn2), a tight junction transmembrane protein, has emerged as a critical regulator of epithelial barrier function and cellular permeability, and critically has significantly upregulated expression in CRC compared to low expression in surrounding tissue. Dr. Singh (MPI) has generated advanced models of CRC growth and progression using colonoscopy guided injection of the tumor cells into the colonic mucosa as well as novel murine models of CRC progression with genetic manipulation of colonic Cldn2 expression. On the other hand, Dr. Mohs (MPI), along with Dr. Singh and collaborators, have extensive experience at developing near infrared fluorescent (NIRF) imaging agents with controlled optical, targeting, and biodistribution properties for image-guided surgery. Most recently, we have focused on developing immunotargeted probes for a wide range of tumors, including GI malignancies. Overall, we reported strong, specific contrast in these studies. Key advantages of this approach for FGS are the availability of antibodies towards tumor-specific antigens and straight-forward NIRF conjugation procedures. Therefore, the goal of this proposal is to design NIRF imaging agents that can target specifically Cldn2 and that can demonstrate the feasibility of NIRF-antiCldn2 antibodies for contrast-enhanced colon cancer imaging using select in vitro and in vivo models. This goal will be accomplished by the following Specific Aims: (1) To synthesize and characterize NIRF anti-Cldn2 conjugates and establish specificity in tumor spheroid models and (2) To evaluate NIRF-antiCldn2 surgical imaging agents using in vivo CRC tumor models. Completion of this research project will identify a NIRF- antiCldn2 conjugate that specifically targets Cldn2 with in vivo evidence that these conjugates can target tumor xenografts in vivo, included an orthotopic model of CRC. These are essential data that are required for further preclinical development under larger funding mechanisms.

NIH Research Projects · FY 2026 · 2026-06

Abstract The advancement of combination antiretroviral therapy (ART) has improved the lives of people living with HIV (PWH) to manageable diseases. However, due to the persistence of viral reservoirs, a cure for HIV remains elusive. Chronological aging is associated with cerebral and extracerebral progressive accumulation of amyloid fibrils, which plays a crucial role in the development of late-onset Alzheimer's and Alzheimer's disease-related dementias (AD-ADRD). More than ten precursor proteins, including transthyretin, amylin, lactadherin, Aβ1-42, α- synuclein, epidermal growth factor-containing fibulin-like extracellular matrix protein 1, semenogelin, and others, are implicated in age-associated amyloidosis. Age-associated amyloidosis results in activation of microglia in the brain, T cells and myeloid cells in the periphery, M1 polarization of macrophages, and creates a systemic chronic inflammatory milieu. In the United States, more than half of all the people living with HIV (PWH) are aged 50 years or older. Additionally, more than 15% of newly HIV diagnosed Americans are over 50 years of age. Therefore, it becomes paramount to understand the impact of amyloid fibril deposition on the reactivation and expansion of lymphoid and myeloid latent reservoirs in older people living with HIV. Conversely, HIV infection and chronological aging are two mutually exclusive comorbid conditions for the development of AD-ADRD. Therefore, it is indispensable to understand the role of HIV in enhancing the deposition of amyloid fibrils and in the development/acceleration of AD–ADRD among older people living with HIV. The commonly used small animal models of AD, not comprehensively recapitulate the heterogeneous manifestations of late onset of AD. On the contrary, rhesus macaques naturally develop age-associated cognitive decline and deposition of amyloid fibrils closely mirrored in humans. This proposal aims to address the knowledge gaps in the field described above. In Aim 1, we will study the impact of age-associated deposition of amyloid fibrils on HIV reservoirs, and in Aim 2, assess the impact of viral infection on the enhancement of chronological aging-associated amyloidosis, and subsequent development of AD-ADRD phenotypes. We anticipate that understanding the aging-associated reactivation and expansion of the viral reservoir will help to provide a more informed approach to designing HIV cure research interventions for older people living with HIV, as well as understanding the role of HIV on the development of late-onset AD-ADRD will aid in the development of customized care modalities, including treatment for comorbid conditions.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract: HIV/AIDS continues to affect nearly 39.9 million people worldwide. Although combination antiretroviral therapy (cART) effectively suppresses viral replication and reduces progression to AIDS, approximately 30–60% of people living with HIV (PLWH) still experience HIV-associated neurocognitive disorders (NeuroHIV), characterized by chronic neuroinflammation and progressive cognitive decline. This persistent inflammatory state is fueled by viral proteins such as Transactivator of transcription (Tat) that continue to be expressed in the brain, lifelong exposure to cART, and high rates of comorbid stimulant misuse, particularly cocaine, which exacerbates neuroinflammation and neuronal injury. Microglia, the resident immune cells of the brain, are central mediators of this process, as excessive activation drives inflammasome signaling, oxidative stress, and synaptic dysfunction. Mechanistically, these effects converge on dysregulated autophagy, lysosomal dysfunction, and impaired iron homeostasis. A critical yet underexplored pathway is ferritinophagy, the selective autophagic turnover of ferritin mediated by nuclear receptor coactivator 4 (NCOA4). When ferritinophagy is disrupted, ferritin accumulates, lysosomal acidification is impaired, and iron overload promotes oxidative stress, ferroptosis, and neuroinflammation. Our preliminary studies demonstrate that HIV Tat, cART, and cocaine cooperatively disrupt lysosomal integrity, impair ferritinophagy, and drive iron-mediated oxidative stress in microglia. Notably, we have identified C381, a novel brain-penetrant lysosomal activator in early clinical development, which restores lysosomal acidification, enhances autophagic flux, and protects against neuroinflammation. Our long-term goal is to develop translational strategies that alleviate neuroinflammation and cognitive decline in PLWH with stimulant use disorder. The short-term goal of this project is to define how HIV Tat, cART, and cocaine disrupt ferritinophagy in microglia and to test whether restoring lysosomal function with C381 can rescue ferritinophagy and mitigate neuroinflammation. Specific Aim 1 will dissect molecular mechanisms of ferritinophagy disruption in vitro and test whether C381 restores iron homeostasis and suppresses downstream inflammatory pathways. Specific Aim 2 will examine the efficacy of C381 in vivo using inducible Tat and EcoHIV mouse models with cocaine and cART exposure and validate ferritinophagy dysregulation in archival macaque and postmortem human brain tissues. Overall, this project will establish ferritinophagy as a mechanistic link between HIV, cART, and stimulant misuse, and neuroinflammation, while testing C381 as a therapeutic strategy. Because C381 is already advancing in clinical development, these studies have immediate translational potential for protecting brain health in PLWH with stimulant use disorder.

NIH Research Projects · FY 2026 · 2026-05

Project Summary The University of Nebraska Medical Center (UNMC) requests funds to purchase a Rigaku HyPix-Arc 100° Curved Hybrid Photon Counting (HPC) Detector to provide state-of-the-art crystallographic X-ray diffraction data collection for the three-dimensional solution of medically important biological macromolecules and their complexes. This instrument for macromolecular crystallography is not currently available at UNMC, any nearby university or in the region. The HyPix-Arc 100° is a unique, curved detector using HPC technology. To provide the best data quality it is essential to measure accurate data with minimal correction. With a curved detector arrangement, diffracted beams arrive at the detector as close to perpendicular to the surface as possible. This prevents unwanted reflection enlargement and minimizes the associated corrections. Additionally, the curvature allows the detector to see a higher theta range than larger flat detectors. This means more reflections collected at the same time under the same conditions for truer measurement with better scaling and data quality whilst also increasing measurement speed for sensitive or unstable samples. This instrument will dramatically improve the quality of X-ray diffraction data we collect to solve crystal structures for basic science and drug development projects. The proposed project is viable because of strong investigator support illustrated by the 15 NIH- funded researchers and their projects on nucleosome assembly, ribonucleoproteins, enzyme catalysis, redox biology, cancer therapeutics, neurodegenerative and infectious disease. Participating investigators are from several departments at UNMC, as well as nearby institutions, such as the University of Nebraska-Lincoln, and Creighton University. Each project will benefit directly from the use of the HyPix-Arc 100° detector: enabling sophisticated experiments and expediting progress on NIH-funded science. These projects enjoy strong technical support in the Eppley Structural Biology Facility (ESBF) that includes well-experienced staff that have worked together with the director for over 23 years promoting all aspects of structural biology research in Nebraska and surrounding states. The proposal also has strong institutional support as evidenced by salary and service agreement support by the Vice Chancellor for Research (Dr. Bayles) and the Director of the Eppley Institute for Research in Cancer (Dr. Sweasy). The detector will be housed in the ESBF and installed on the right port of our Rigaku FRE+ ultrahigh-intensity rotating anode X-ray generator. The ESBF is the only structural biology facility in the region and gives easy access for data collection to all interested researchers. The combination of strong user interest, significant research projects, technical expertise, administrative experience, and a solid long-term plan will ensure successful implementation and extensive use of the HyPix-Arc 100° detector.

NIH Research Projects · FY 2026 · 2026-05

This is a new multiple-PI R01 application to test the innovative approach of targeting hepatic cell signaling mechanisms involved in alcohol-related colorectal liver metastasis (CRLM). The aberrant use of alcohol associates with the development of cancers including colorectal cancer (CRC), the second leading cause of death in the United States. Most CRC deaths are due to metastases to the liver, the foremost site of secondary organ involvement. Despite advancements in screening and treatments for primary CRC, few effective options are available for liver metastatic disease. In search of targetable mechanisms involved in CRLM, research has shown a correlation between the expression of the tumor antigen, carcinoembryonic antigen (CEA), and poor outcomes of CRC, especially in patients with an alcohol use history. CEA is overexpressed in metastatic CRC cells and stimulates macrophages in the host liver to produce inflammatory factors that promote CRLM. CEA signaling in alcohol-affected MΦs leads to the production of metastatic factors and the activation of hepatic stellate cells (HSC) resulting in matrix remodeling for CRC cell expansion in the liver. We hypothesize that pre- existing alcohol-associated liver disease drives hepatic macrophages toward a CEA-responsive, prometastatic phenotype that can be targeted by novel nanoparticle (NP) formulations. To investigate this, three specific aims are proposed to 1) develop and optimize novel NPs for maximal inhibition of alcohol-related macrophage activation by CEA, signaling via miR-155, and HSC transformation via inhibition of the chemokine receptor CXCR4; 2) characterize the effectiveness of the anti-CRLM NPs and the contribution of metastasis-promoting MΦs using ex vivo and in vivo assessments; and 3) to perform transcriptomic analysis to determine the intrahepatic MΦ gene signature, localization, and prometastatic properties during alcohol-associated CRLM. The successful completion of this work will define the effectiveness of novel therapeutic strategies aimed at reducing or eliminating CRC metastatic burden in the liver. Also, we will determine hepatic cell involvement during alcohol-mediated exacerbation of CEA signaling and advancement of CRLM and underlying mechanisms of action of anti-CRLM nanoparticles. This is a clinically relevant topic and priority area of investigation which has the potential to significantly impact the clinical care for individuals with liver metastasis of colorectal cancer. Overall, this characterization of targetable macrophage populations and tumor-promoting signaling pathways can lead to translational breakthroughs in the field, and next-step evaluations to effectively reduce the severity of metastatic disease.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract My professional career goal is to conduct independent research in life sciences defining complex biological systems, including and specifically alcohol-associated liver disease (ALD). ALD is a progressive disease that can lead to irreversible and fatal liver damage in the form of inflammation, fibrosis, cirrhosis, and cancer. Unfortunately, therapeutic treatments for ALD are limited. This is in part due to the lack of understanding of ALD progression at the cellular level. The onset of ALD is represented by a reversible steatotic liver stage where lipid droplets (LDs) excessively accumulate within hepatocytes, the highly metabolizing cell type of the liver. This observation suggests chronic alcohol consumption prevents the breakdown of LDs. Previous studies have shown alcohol disrupts endocytic vesicle trafficking proteins, including Rab5, and LD catabolism; however, these cellular mechanisms of perturbation are poorly understood. Therefore, this proposal aims to elucidate the molecular mechanisms in which alcohol affects Rab5 and endocytic trafficking during ALD progression. Preliminary studies show alcohol disrupts Rab5 subcellular localization without affecting Rab5 activity. Altogether, the central hypothesis is alcohol disrupts Rab5 prenylation, a post-translational modification, to drive its altered localization. This will be tested by two specific aims. Aim 1 will define the impact of alcohol on Rab5 subcellular localization and remodeling of the endosome proteome. Aim 2 will determine the impact of alcohol on global changes in protein prenylation (prenyl-proteome) including Rab5. To accomplish these aims, I will acquire training in confocal microscopy and mass spectrometry. This new training will broaden my current skillsets regarding yeast cell culture and genetics, small molecule trafficking and signaling, the use and design of fluorescent biosensors, and inter-organelle communication. My strong advisory team, including my sponsor and co-sponsor Dr. Micah Schott and Dr. Carol Casey, respectively, one collaborator, and five other faculty have significant expertise in the proposed topics and the desired training objectives. The microscopy and mass spectrometry core facilities, and research environment at UNMC will enhance this research and training. Together, I will have the expertise and training needed to successfully accomplish these aims. The results gained from the proposed research will provide a mechanistic understanding of disrupted endocytic trafficking by alcohol in ALD.

NIH Research Projects · FY 2026 · 2026-05

Colorectal Cancer (CRC) is the third most common cancer in men and women. While the 5-year survival rate for localized CRC is 91%, the survival rate is less than 14% for patients with disseminated disease. With few exceptions, metastatic CRC is incurable, largely due to resistance to available therapies. As a result, CRC is the second leading cause of cancer death in the U.S.A. These statistics highlight an urgent clinical need for develop- ment of effective treatments for patients with advanced CRC. Aberrant cap-dependent mRNA translation initia- tion (CDT) has emerged as an attractive target for the clinical management of CRC based on its key role in supporting selective synthesis of crucial oncogenic drivers such as growth factors, cell cycle regulators, anti- apoptotic proteins, and Myc. A key negative regulator of CDT is 4E-BP1 which blocks the formation of the translation initiation complex, eIF4F, by stochiometric binding to eIF4E. 4E-BP1 acts as a nexus for aberrant upregulation of CDT by oncogenic signaling pathways that drive CRC (e.g., Wnt/β-catenin, RAS/RAF/ERK, PI3K/AKT/mTOR). These pathways inactivate 4E-BP1 by (a) inhibitory phosphorylation, and/or (b) reducing the 4E-BP1:eIF4E ratio through upregulation of eIF4E or downregulation of 4E-BP1. Inactivation of 4E-BP1 is not only critical for the transformed phenotype but is also a major mechanism underlying failure of targeted therapeutics, including inhibitors of mTOR and the ERK pathway, which rely on inhibition of CDT for their antitumor effects. Thus, restoration of 4E-BP1 holds promise for sensitization of CRCs to a broad range of targeted agents. This proposal builds on our finding that a subset of protein phosphatase 2A (PP2A) heterotri- mers are potent regulators of 4E-BP1 and CDT. Our group has developed first-in-class Small Molecule Activators of PP2A (SMAPs) that activate a subset of B56-PP2As for tumor suppression in CRC and other cancer types. A lead SMAP (EV440/SW-3431) is in clinical development by Springworks Therapeutics, with phase 1 clinical trials anticipated in early 2026. We have discovered that SMAPs orchestrate a PP2A-dependent translation repressive program in CRC cells by (a) activating 4E-BP1 through hypophosphorylation, and (b) upregulating 4E-BP1 levels, even in tumors that lack 4E-BP1 expression. These findings support activation of specific PP2A(s) as a powerful strategy for restoration of translation control in CRCs. We hypothesize that 4E-BP1 translation repressive function can be restored in CRC cells by activation of specific B56-PP2A phosphatase(s) for inhibition of translationally regulated oncogenic drivers, tumor suppression, and sensitization to targeted therapies. Three Aims are proposed: (1) Define the B56-PP2A→4E-BP1 signaling axis in colorectal cancer, (2) Explore 4E-BP1- dependent antitumor effects of B56-PP2A activation in colorectal cancer cells, and (3) Determine the potential of PP2A-mediated restoration of 4E-BP1 function for colorectal cancer therapy. Proposed studies will define mechanisms of translation control in CRCs and explore a novel antitumor strategy, i.e., PP2A-mediated restora- tion of active 4E-BP1, for managing advanced CRC in patients.

NIH Research Projects · FY 2026 · 2026-05

ABSTRACT According to the UNAIDS global estimates for 2023, there are a total of 1.4 million adolescents living with HIV. Adolescence is a very sensitive period for social development and any negative experiences such as psychosocial stress can significantly increase the severity of psychiatric and substance use disorders (SUD). This problem is further compounded in HIV+ adolescents due to the associated stigma, negative attitudes, and prejudice in the society. Social defeat (SD)- a model that mimics bullying in humans and is considered a relevant animal model of psychosocial stress in defeated individuals. Adding another layer of complexity is the emerging trend in polysubstance use (PSU) that typically involves the simultaneous use of an opioid along with a stimulant and in recent years, this problem has escalated into a nationwide epidemic. Two studies from our group mimicking PSU via co-exposure of methamphetamine (MA) and the prescription opioid oxycodone (Oxy) revealed alterations in the synaptic proteome including pathways associated with mitochondrial function. Also, our preliminary studies using the HIV transgenic rat (Tg) has revealed increased levels of stress, inflammatory markers and mitochondrial dysfunction in adolescent HIV-Tg rats after SD. Alterations in mitochondrial function is a well-known facet during HIV infection and drug abuse induced CNS dysfunction. However, molecular mechanisms associated with perturbing mitochondrial function after SD in HIV+ adolescents and PSU have not been explored. In this R21, we will elucidate the role of a novel class of extracellular vesicles (EVs) released from mitochondria termed “mitovesicles” and their underlying mechanisms in inducing CNS dysfunction in HIV+ adolescents with PSU after SD. Based on our preliminary studies that revealed increased mitovesicles sizes in the HIV-Tg rats, our central hypothesis is PSU in HIV+ adolescents after SD dysregulates mitovesicle cargo dynamics and exacerbates neuronal injury. Under Aim 1 we will test if PSU in socially defeated HIV+ adolescents dysregulate mitovesiclce dynamics and impair mitochondrial function. In Aim 2, we will characterize molecular mechanisms associated with perturbed mitovesicles dynamics and impaired mitochondrial function elicit higher synaptic injury with PSU in socially defeated HIV+ adolescents. Completion of this study will help expand our knowledge on unraveling molecular mechanisms contributing to CNS damage in a specific subset of vulnerable individuals: HIV+ adolescents exposed to psychosocial stress and with PSU. Notably, the identification of mitovesicle specific protein cargo signatures will fuel future mechanistic studies including developing them as potential therapeutics.

NIH Research Projects · FY 2026 · 2026-05

ABSTRACT Amino acid substitutions in the exonuclease domain of DNA polymerase ε (POLE) cause cancers with extremely high mutation burdens. POLE-mutant status is associated with improved patient outcomes due to high neoantigen production and an enhanced anti-tumor immune response. The ability to recognize driver variants is critical for designing personalized treatment strategies. Mechanisms of ultramutation that defines good prognosis are also of high interest. The long-term goals of our research are to understand the mechanisms of POLE-linked ultramutation and improve the detection of functionally significant variants in patients. Our laboratory experimentally validated 15 of 17 currently known POLE drivers. We also deciphered the mechanisms through which amino acid changes at the protein-DNA interface in the exonuclease domain cause ultramutation. However, much remains to be explored in this relatively young field. Mechanisms remain unclear for other categories of POLE driver mutations, which include a highly prevalent pathogenic variant V411L. Further, we recently discovered that changes in the DNA polymerase domain of POLE can also drive ultramutation in tumors, constituting another poorly understood group of drivers. There is a paucity of experimental models for assessing the consequences of variants in the context of human cells and proteins. Methods to determine POLE status unambiguously in the clinical setting are also lacking. The proposed research will fill these gaps. We will use novel genetic, biochemical, and computational tools and a broad arsenal of experimental models, including “humanized” yeast, POLE-mutant human cells, knock-in mouse lines, and clinical samples to facilitate this research. In Aim 1, we will develop new, improved approaches to identify POLE tumors and disambiguate variants of unknown significance. In Aim 2, we will characterize novel drivers affecting the DNA polymerase domain of POLE. In Aim 3, we will define the mechanism of tumorigenesis for the highly recurrent V411L variant. This research will advance the mechanistic understanding of POLE-driven tumorigenesis and help address current clinical needs.

NIH Research Projects · FY 2026 · 2026-04

Project Summary/Abstract West Nile virus (WNV) is the most consequential arthropod-borne virus (arbovirus) in the US, and due to the lack of effective treatments or vaccines, preventing human exposures to infectious mosquitoes is key to controlling transmission. Mosquito-based surveillance for WNV in the US is robust and provides policy makers information on the human risk of WNV infection in near “real-time” in the form of the Vector Index (VI), a measure of the abundance of infected mosquitoes in a given area. Human cases are nearly always preceded by an increase in the number of mosquitoes testing positive for WNV, and thus an increase in the VI, weeks prior to onset. However, an increase in the VI is not always followed by the detection of human cases. These observations suggest the accuracy of the VI can be improved. The VI is calculated by determining the number of mosquito pools that test positive for WNV, typically through standard RT-qPCR assays. While these assays are inherently quantitative, the outputs are interpreted in a binary fashion, either the presence or absence of WNV RNA. Quantity of WNV RNA from mosquito surveillance pools, as measured by cycle threshold (CT) values, varies dramatically and is not considered when calculating or interpreting the VI. The objective of this proposal is to determine how the variability in WNV CT values from mosquito surveillance pools is associated infectiousness (i.e. the point at which a mosquito can transmit WNV) in mosquitoes and to use these data to refine the VI. The central hypothesis is that WNV CT values are a reliable predictor of infectiousness in mosquitoes, and therefore useful indicators of human risk of infection. This hypothesis will be tested in two independent and complimentary aims. Aim 1: Identify the biological underpinnings of the observed variability in WNV RNA quantities from mosquitoes. This aim will make use of a robust experimental design to expose mosquitoes to i) a range of WNV titers via lab-derived bloodmeals and ii) viremic blood collected through the course of experimental infection in birds to determine how these factors affect WNV growth, time to infectiousness, and titers in saliva. Importantly, the results of these experiments will make the explicit connection between whole body CT values (what is measured in WNV surveillance) and the ability of mosquitoes to transmit WNV. Aim 2: Incorporate WNV CT values generated from mosquito surveillance into measures of human risk. This aim will result in two biostatistical modeling strategies that incorporate WNV CT values produced during routine mosquito-based surveillance into measures of the VI. The accuracy of these models to predict the timing and magnitude of human WNV cases will be determined using a robust retrospective dataset of over a decade of human and mosquito WNV surveillance data from Nebraska and Colorado. Taken together, this R21 is addressing a significant knowledge gap in arbovirus surveillance in the US by using innovative experimental and biostatistical approaches to identify the biological underpinnings of WNV RNA variability in naturally infected mosquitoes and to use these data to better estimate human risk and inform control campaigns.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY The subcellular localization of a biomolecule often reveals its functional significance. RNA has typically been confined to the cytoplasm and nucleus, where it is critically involved in numerous biochemical processes. Recently, glycosylated RNAs (glycoRNAs) have been discovered on the cell surface of various cell types, raising important questions about their functional relevance, mechanisms of action, and the trafficking pathways that maintain their dynamic equilibrium on the cell surface. The long-term goal of this proposal is to discover and define the functional mechanisms of cell surface RNAs in endocytosis and pathogen endocytic trafficking. Preliminary evidence suggests that cell surface RNAs are critically required in human papillomavirus (HPV) infection. This MIRA proposal is firstly focused on deciphering the functional roles of cell surface RNA in HPV endocytic trafficking. Here a combination of advanced imaging strategies, biochemical techniques, and genetic analysis will be used to dissect and determine the specific step in HPV cell entry that is regulated by cell surface RNAs. We are also exploring the mechanisms underlying this process by leveraging recent discoveries from my lab, including the identification of the cell surface RNA binding protein (RBP) DDX3X, a potential recognition site for cell surface RNA-DDX3X clustering on the HPV cell-penetrating peptide (CPP), and glycoRNA transcripts identified through RNA sequencing. Lastly, we are investigating the trafficking modes utilized by cell surface RNAs, which are critical for their cell surface presentation and functions. Collectively, this research addresses fundamental questions in cell membrane trafficking and provides valuable insights into the recently discovered cell surface RNAs. It may also introduce a new concept in which cell surface RNAs and RBPs cluster to form an endocytic receptor complex involved in pathogen cell entry. While many exciting questions lie beyond the scope of this MIRA proposal, the findings could stimulate broad interest, offer proof-of-concept frameworks, and develop techniques that enhance our understanding of the role of cell surface RNAs in endocytosis and pathogen entry mechanisms.

NIH Research Projects · FY 2026 · 2026-04

Prosthetic joint infection (PJI) is a complication following arthroplasty that occurs in approximately 1-3% of patients, with ~50% of cases caused by methicillin-resistant Staphylococcus aureus (S. aureus). Biofilm formation evolves as bacteria multiply, producing a complex extracellular matrix that creates a heterogeneous population with unique metabolic properties. S. aureus is metabolically flexible, allowing it to colonize various niches and establish chronic infections. Bacterial-derived metabolites play an important role in biofilm infection, which is reflected by the fact that several tricarboxylic acid cycle (TCA) intermediates are critical for biofilm development. Our laboratory has characterized an in vivo mouse model of PJI and identified macrophages (Mφs), anti-inflammatory granulocytic myeloid-derived suppressor cells (G-MDSCs), and polymorphonuclear cells (PMNs) as the predominant leukocyte infiltrates, all of which are also present in human PJI. Previous work from our group has characterized several metabolites that are important for promoting S. aureus persistence during PJI; however, it is unclear how S. aureus adapts in response to leukocyte exposure to regulate its metabolism to promote biofilm formation and establishment. Our novel bacterial single-cell RNA sequencing (Bsc-RNA seq) approach has revealed that Mφs, G-MDSCs, and PMNs elicit distinct metabolic changes in S. aureus biofilm, namely, the expression of genes involved in citrate metabolism (gltA, acnA, and icd). While the role of AcnA has been examined in a S. aureus skin and soft tissue infection model, its role, along with the other citrate enzymes (GltA and Icd) in biofilm development under leukocyte pressure and during PJI, remains elusive. The overarching hypothesis of this fellowship is that S. aureus citrate metabolism is upregulated to adapt to leukocyte pressure and promote biofilm development and pathogenesis. The role of citrate metabolism in biofilm development and S. aureus pathogenicity will be examined in the following Specific Aims. 1) Investigate the role of S. aureus biofilm citrate metabolism under leukocyte pressure and 2) Elucidate the role of citrate metabolism on S. aureus pathogenesis in PJI. The results from this fellowship will provide new insights into S. aureus metabolic adaptations under leukocyte pressure and within the PJI niche that could represent future therapeutic targets.

NIH Research Projects · FY 2026 · 2026-02

Peripheral artery disease (PAD) is a progressive condition marked by the narrowing and blockage of arteries supplying the legs, often leading to debilitating leg pain and significant walking impairment known as claudication. While standard therapies exist, their effectiveness is limited, and there remains a critical need for treatments that enhance limb perfusion and function while reducing reliance on costly interventions. Recent clinical trials have suggested that stem cell therapy may hold promise for PAD treatment, yet results have been mixed, with ongoing barriers regarding the optimal cell type, delivery method, and therapeutic targeting. In this proposal, we overcome these barriers by using an autologous cellular preparation (adipose stromal vascular fraction, or SVF), and by targeting the inflow collateral vessels and employing a novel access and delivery strategy to enhance perfusion. We have developed and validated a minimally-invasive porcine model of hindlimb ischemia (percutaneous catheter-based coil occlusion of the iliofemoral and popliteal arteries) which recapitulates key aspects of human PAD and can be a platform for PAD therapy development. We have demonstrated that transvenous periarterial administration (around the porcine aortic trifurcation) of SVF increases long-term arterial inflow to the ischemic hindlimb and improves treadmill performance with respect to sham-treated ischemic hindlimbs. The objective of our proposal is to compare regenerative, cell-based regimens for PAD treatment (including SVF and SVF-derived exosomes) in a clinically relevant animal model of hindlimb ischemia whose size, anatomy, physiology, and comorbidities closely mirror those of human PAD patients. Our central hypothesis is that: (i) delivery of SVF into the peri-arterial region of the aortic trifurcation, in our porcine hindlimb ischemia model, will increase arteriogenesis and improve hemodynamic and functional endpoints more effectively than either intra-arterial SVF delivery or sham treatment; and (ii) SVF vs. SVF-derived exosomes, delivered peri-arterially in the same model, will provide equivalent benefits on the same endpoints. We will test this central hypothesis in three Specific Aims: Aim 1 will compare the effect of transvenous/peri-arterial (aortic trifurcation region) SVF delivery vs. intra- arterial SVF delivery on arteriogenesis, hindlimb perfusion, treadmill performance, and histological endpoints in our porcine model of hindlimb ischemia; Aim 2 will compare the effect of transvenous/peri-arterial (aortic trifurcation region) delivery of SVF vs. exosomes derived from SVF on arteriogenesis, hindlimb perfusion, treadmill performance, and histological endpoints; and Aim 3 will investigate the mechanisms by which SVF or exosomes delivered to the retroperitoneum around the aortic trifurcation drive arteriogenesis. Successful performance of this research proposal should lead to development of arterial inflow-enhancing therapies which would be useful in PAD patients, particularly ones who are not fit, not appropriate, or not willing to undergo a major revascularization procedure.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Research on factors influencing the early progression of Alzheimer’s disease (AD) has primarily focused on persons with dementia (PWD), such as their genetics. Most PWDs live within their communities, and many maintain close connections with their spousal caregivers (CGs). Relationship science research has shown that close connectedness to family, particularly spouses, benefits health; however, the influence of interpersonal connectedness between PWDs and spousal CGs on early AD progression has been overlooked. Family is part of the community, and the influence of PWD-CG connectedness on AD progression may be moderated by the dyad’s connectedness to their communities (e.g., receiving social support), which can differ between rural and urban areas. Our central hypothesis is that stronger PWD-CG connectedness is associated with slower AD progression. In a 2-year longitudinal study, we will recruit 71 urban and 71 rural co-living spousal care dyads where care recipients are in the early stages of late-onset AD. Laboratory measures of PWD- CG connectedness (e.g., observations of synchrony in positive emotions during face-to-face interactions) and AD progression (e.g., increased clinical symptoms and reduced brain volumes) will be collected at baseline and at a 2-year follow-up. Real-world wearable measures (i.e., the dyad’s in- home physical proximity and movement synchrony during daily social interactions for a 2-week period) and questionnaire measures of PWD-CG connectedness will be collected every 6 months throughout the study. Analyses will adjust for known risk factors for AD progression (e.g., APOE-ε4) and other confounding factors (e.g., medications, personality). Using a multi-method design, at the end of the 2-year follow-up period, we will also conduct qualitative interviews with the dyads to integrate their explanations of the quantitative findings into the study's outcome interpretation. Analyses will be conducted to determine how PWD-CG connectedness influences early AD progression (Aim 1), how communities (urban and rural) influence PWD-CG connectedness and its impact on early AD progression (Aim 2), and the value of measuring PWD-CG connectedness using wearables (Aim 3). Findings will significantly advance the understanding of social and psychosocial factors in disease mechanisms and protective factors for AD. The findings will help identify early predictors, digital markers, and novel interventions targeting PWD-CG connectedness and, more broadly, family and community connectedness, to mitigate early AD progression.

NIH Research Projects · FY 2025 · 2025-09

Abstract Glioblastoma (GBM), the most common malignant primary brain cancer in adults, has an average survival of one year. Treatment includes maximal safe resection, followed by chemoradiation and adjuvant temozolomide (TMZ), the latter only increasing median survival by 2.5 months. Moreover, most targeted therapy trials have been unsuccessful due to activation of tyrosine kinase receptors and relative blood-brain barrier (BBB) impermeability. Thus, a pressing need remains to find a more effective therapy. This study builds on preliminary data generated using Connectivity Map (CMap), developed by the BROAD institute to identify drugs for repurposing based on cancer’s genetic profile. By analyzing 99 GBM and 38 adjacent normal samples from 4 datasets, CMap identified histone deacetylase (HDAC) inhibitors as top candidates. Database analysis using GEPIA identified HDAC1 and HDAC2 as the most upregulated and HDAC11 as the most downregulated HDACs. Thus, we selected the BBB permeant PCI-24781/abexinostat due to its specificity for GBM gene signature- specific HDACs (inhibiting HDAC1 and HDAC2, but not HDAC11, or class IIa HDACs) to evaluate. In preliminary studies, as compared to other pan HDAC inhibitors, PCI-24781 induced significantly greater apoptosis and downregulated DNA repair machinery (CHK1, RAD51, and MGMT) in GBM cell lines in vitro. Further, PCI-24781 efficiently decreased the tumor burden in orthotopic murine models in combination with TMZ compared to vorinostat with TMZ and enhanced survival. From this, we hypothesize that inhibiting class 1 HDACs with PCI- 24781 will enhance the efficacy of TMZ in GBM by targeting DNA repair machinery. To test this hypothesis, we propose two specific aims: In Aim 1, we will evaluate the efficacy of PCI-24781 with TMZ in in vivo GBM models. Murine GBM organoids, genetically engineered GBM mouse models, and patient-derived xenografts will be used to evaluate BBB permeability and to measure toxicity. Therapeutic efficacy and survival will be recorded for each group of mice treated with PCI-24781 and TMZ singly and in combination. We will analyze the effect of combination therapy on DNA repair machinery proteins by IHC in resected tumors and will also perform RNA- seq and Omni-ATAC-seq analyses to identify additional pathways and chromatin accessibility, respectively, impacted by PCI-24781. In Aim 2, a phase 1 clinical trial will evaluate toxicity and determine the MTD of PCI- 24781 with TMZ in recurrent high-grade glioma patients. We will analyze patient exosomes to demonstrate protein acetylation and BBB permeability. Together, these aims will elucidate mechanisms for synergy between and tolerance of PCI-24781 with TMZ in GBM. Combining PCI-24781 with TMZ will successfully overcome TMZ resistance, negatively impacting tumor growth and recurrence and effectively improving the survival of GBM patients.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Aberrant activation of receptor tyrosine kinases (RTK) drives or enables tumorigenesis, metastasis, and therapeutic resistance across cancers but RTK targeting is only successful in few of these. IGF-1R provides a key oncogenic axis in Ewing Sarcoma (EWS), the second most common malignant bone tumor of children and young adults with a 5-year survival rate of only 39% once metastasized. Yet, clinical trials showed limited responses to IGF-1R inhibitors in EWS. An essential cancer hallmark is the metabolic rewiring with enhanced accumulation of glucose and its aerobic glycolysis (the Warburg effect). This helps reallocate glucose-derived metabolites for macromolecular synthetic needs of proliferative tumor cells and away from use in bioenergetics. Increased glucose uptake (using FDG-PET scanning) is a mark of EWS patient tumors and correlates with worse outcomes. Yet, knockdown (KD) of the EWS driver oncogene EWS-FLI1 led to enhanced glucose uptake and glycolysis, suggesting other molecular pathways counter the EWS-FLI1 effect to inhibit glycolysis. We identified EHD1, a member of the EPS15-Homology Domain-containing (EHD) protein family, as a required element of the EWS tumorigenesis and metastasis. Mechanistically, EHD1 promoted the intracellular traffic of IGF-1R to elevate its cell surface expression and signaling. Our new data demonstrate that EHD1 overexpression upregulates glucose uptake, essential for glycolysis, in an IGF-1R dependent manner. Metabolomics analyses demonstrated that EHD1-KO, which genetically mimics IGF-1R inhibition, in EWS cell models led to a switch to glutamine- dependent maintenance of TCA cycle, in the process of glutamine anaplerosis. Glutaminase 1 (GLS) or GLS2 convert glutamine to glutamate for entry into metabolic pathways. GLS is universally pro-oncogenic while GLS2 is context dependent. We found that EWS cells only express GLS. GLS inhibition is untested in EWS but effective in other cancer models. GLS inhibitor CB-839 (Telaglenastat) was found safe in phase 1 trials and is in phase 2 trials in prostate cancer (NCT04824937). We found CB-839 to inhibit EWS cell proliferation and migration at nanomolar IC50s, and to be synergistic with Linsitinib, an IGF-1R inhibitor in advanced clinical trials. We hypothesize that a metabolic switch to glutamine dependence sustains EWS tumorigenesis and metastasis in the face of IGF-1R inhibition, and thus concurrent targeting of IGF-1R and GLS will provide an effective targeted therapy approach for EWS. We propose complementary genetic and pharmacologic studies using cell line and Patient-derived xenograft (PDX)-derived tumor organoid models of EWS to assess if GLS is a valid therapeutic target in EWS (Aim 1) and if its co-targeting with IGF-1R inhibitors will be additive or synergistic (Aim 2). Our studies evaluate a novel hypothesis, use state of the art and innovative approaches, and explore a co-targeting approach not previously tested. Success of our studies will nominate IGF-1R and GLS inhibitors as a novel and safe combinatorial targeted therapy for EWS. The novel principle of IGF-R plus GLS inhibition may be extendable to other cancers (including NSCLC, breast, and thyroid) with an overactive EHD1-RTK axis.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Background: The number of women living with human immunodeficiency virus type 1 (HIV-1) getting pregnant while on antiretroviral therapy is steadily rising. This is due to the growing availability of antiretroviral drugs (ARVs) at affordable access worldwide. Each year, more than a million women LWH have given birth while on ART. Although ART has significantly reduced the rate of vertical HIV-1 transmission to less than 1%, risks of adverse events on fetal outcomes linked to in utero ARVs exposure remain a major concern. Studies showed that HIV-1-exposed uninfected (HEU) children are at increased risk for preterm delivery, infectious morbidity, mortality, immune abnormalities, and impaired growth or neurodevelopment; yet there are gaps in understanding of how in utero ARVs exposures may impact placental development, the key regulator of fetal growth. One of the major reasons behind this limitation include co-morbidities complicating the interpretations. There is a substantial knowledge gap about the effects of comorbid ARV’s and substance use on placenta. Several addictive agents are commonly used by pregnant woman LWH, however, globally nicotine exposure through smoking or tobacco use remains as one of the major co-morbidities. Smoking is an independent risk factor for adverse fetal outcomes, such as preterm delivery, low birth weight, and intrauterine growth restriction. However, whether and how, ARVs and nicotine together produce synergistic effect to exacerbate adverse effects on placental development affecting pregnancy outcomes is unknown. Objective: Uncover how co-exposure of nicotine exacerbate ARVs-linked adverse effects on placenta development and employ long-acting (LA) delivery means to alleviate the impact of co-morbidity by attenuating ARVs-linked adverse effects. For ARVs, the focus will be on commonly prescribed and first-line integrase strand transfer inhibitors (INSTIs). Preliminary data: We observed that dolutegravir (DTG) reduced MMP-2 activity and HIF-1α protein accumulation under both normoxic and hypoxic conditions in cultured trophoblasts, reducing migration abilities. Moreover, nicotine further exacerbated the effect of DTG on HIF-1α protein accumulation. DTG-induced abnormalities in these proteins influenced impairment in placenta vasculature development in rodent model. Hypothesis: Synergistic effects of INSTI and nicotine on MMPs and HIF-1α impairs placental morphology and vasculature development. Research Strategy: (1) Assessment of effects of co-exposure of INSTIs and nicotine on trophoblasts functions and associated alterations in global proteomic profile; (2) Determine synergistic impact on placental development in a rodent model; and (3) Evaluate injectable long-acting delivery means for protective outcomes. Expectations: Nicotine exacerbates adverse impact of INSTIs on placenta development and that long-acting delivery approach will attenuate the impact of co-morbidity by attenuating INSTIs’ exposure to placenta.

NIH Research Projects · FY 2025 · 2025-09

Abstract In age-related cataracts (ARC), increasing levels of oxidative stress due to dysfunction of Nrf2 (NFE2-related factor)-mediated antioxidant defense is widely recognized, but much remains unexplored. Nrf2 is known to extend health/life span by maintaining redox homeostasis. However, with advancing age Nrf2 antioxidant function deteriorates, resulting in etiopathobiology. A better understanding of the molecular regulation of the age-dependent dysfunction of Nrf2-antioxidant pathway and its link to aging-related pathobiology and thereby, finding the way(s) to ameliorate the Nrf2-antioxidant pathway can offer an opportunity to prevent ARC. Recently, our in-vitro/in-vivo/ex-vivo studies with aging lens/lens epithelial cells (LECs) discovered that the anti- aging drug, Metformin (Met) is efficacious in activating the deteriorated Nrf2/ARE (antioxidant response element) antioxidant pathway via AMPK activation, resulting in lens/LECs protection. Notably, we identified that Nrf2 and antioxidant genes, like Prdx6, can be synergistically activated via Bmal1 (brain and muscle Arnt-like protein 1)/E-Box and Nrf2/ARE activities. Pursuing this line, we found that Met negatively regulates both the expression/activity of BTB and CNC homology 1 (Bach1), an inhibitor of Nrf2 at ARE site(s), and revives Nrf2/ARE pathway. Preliminary data also showed that Bmal1 or Nrf2 deficiency and increase of Bach1 levels in aging lenses/LECs attenuate the Nrf2/ARE pathway. We envisioned that with aging, within the eye lens, increased oxidative stress due to deterioration of Bmal1/Nrf2/ARE pathway creates a toxic milieu promoting disease state, ARC. The molecular pathways underlying this process, however, remain elusive. Using Met as a tool, the overall goal of this proposal is to determine the mechanisms underlying dysregulation of the AMPK/Bmal1/Nrf2/ARE axis gene networks axis in aging eye lenses/ARC, and to examine the transcriptional reprogramming of this axis and its contribution in abating aging pathobiology in response to Met by using state- of-the-art RNA-Seq with biochemical approaches. Our initial data using each of these factors also showed that; (i) Bach1 overexpression inhibits Nrf2 activation of antioxidants transcription, while Met treatment revives the process; (ii) Cotreatment with an AMPK inhibitor blocks Met activation of Bmal1/Nrf2/ARE pathway; (iii) Met instillation in aging mice eye, retards the lens pathobiology in-vitro/ex-vivo/in-vivo. These promising data support the hypothesis that the dysregulation of Bmal1/Nrf2 gene networks can be revived by Met. We will test the hypothesis via three specific aims: (1) Uncover the causes and the mechanisms of Bmal1/Nrf2 antioxidant pathway dysregulation in aging/oxidative stress in vitro. (2) Determine the mechanism(s) of Met- mediated revival of Bmal1/Nrf2/antioxidant pathway and other target genes, and attenuation of lens opacity in- vitro. (3) Determine the mechanisms underlying Met-mediated activities in preventing lens opacity in a mouse model(s) of ARC in-vivo. The mechanisms of pathological signaling and role of Met uncovered in this proposal should have a translational impact on increasing aging-related blinding diseases and improving public health.

NIH Research Projects · FY 2025 · 2025-09

Project Summary I am a Research Manager in Dr. Surinder Batra’s lab at the University of Nebraska Medical Center (UNMC). Dr. Batra’s laboratory is engaged in basic and translational research in pancreatic cancer and uses several genetically engineered mouse models (GEMMs) to study molecular mechanisms, biomarkers, and therapeutic modalities. My expertise is in providing unique resources to Drs. Hollingsworth, Jain, Ouellette, Ponnusamy, Singh, and others research on pancreatic cancer (PC). I am also the Comparative Medicine designated Laboratory Animal Coordinator for supervising Batra Lab’s animal research. My role has been critical in developing all the GEMMs for PC. Dr. Batra has been working on pancreatic cancer to study the role of mucin (MUC4, MUC5AC, and MUC16) and non-mucin genes (C1GALT1, PD2, CXCR2, NCOA3, and NGAL) in disease pathobiology and evaluating their utility for early detection and therapy. Specifically, Dr. Batra’s laboratory is funded to investigate the molecular mechanism of pancreatic cancer progression and metastasis, evaluate biomarkers, and test therapeutic agents. This R50 award will support my efforts towards four significant aspects of Dr. Batra’s research program, funded by P01, two U01s, and several R01 Awards from NCI. These projects are focused on: a) understanding MUC16 mucin-mediated molecular mechanisms for pancreatic cancer metastasis; b) investigating the role of truncated O-glycans on cancer-associated glycoproteins (membrane-tethered mucins and stemness markers); c) develop and evaluate MUC4- targeted near-infrared (NIR) probes for optical surgical navigation of PC; d) validation of biomarkers for risk prediction and early diagnosis of Pancreatic Adenocarcinoma; e) examining mucin-based biomarkers to improve early cancer detection. I am involved in various aspects of the Unit Director’s research program, including 1) animal model development, 2) experimental therapeutics, and 3) biomarker research. I have been deeply involved in optimizing the production of in-house generated therapeutic antibodies using advanced mammalian expression systems and bioreactors (compatible with GMP scale antibody generations) and assessing their efficacy in pancreatic tumor xenografts. My major focus has also been developing and maintaining KC and KPC GEMMs of PC and further engineering these lines for the overexpression/knockout with project-specific genes (MUC4 and MUC16). My biomarker research involvement includes assays and reagent development, biospecimen processing archiving and distribution, and data coordination. I have spearheaded efforts to employ innovative approaches for experimental therapeutics (organoid models, Live cell imaging, antibody efficacy) and biomarker research (Digital Droplet PCR and Multiplex assays). My continued involvement in PC research under the Unit Director’s supervision can lead to novel diagnostics approaches and develop innovative antibody modalities for PC treatment.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Rheumatoid arthritis (RA) is a common autoimmune disease characterized by joint inflammation and damage as well as systemic manifestations including cardiovascular disease that lead to premature mortality. People with RA suffer up to a 2-fold increased risk of heart failure (HF), among the most burdensome chronic conditions and the most common cause of hospitalization in the U.S. This heightened risk of HF in RA, including HF subtypes with preserved and reduced left ventricular ejection fraction, has not improved despite substantial RA treatment advances over the last two decades. The mechanisms propagating HF risk in RA are poorly understood, prohibiting the development of effective strategies to improve HF-related outcomes in RA. Given the rising prevalence of HF in an aging population and the concerning persistence in RA-related HF risk, a critical need exists to identify unique drivers of HF development in RA to serve as priority targets for the design of HF prevention and management strategies in this high-risk patient population. The overall objective of this study is to identify molecular determinants and epidemiologic mediators of early myocardial dysfunction and clinical HF in RA, while completing a robust training program in prospective cohort development, cardiac phenotyping, and causal inference methodology. The central hypothesis is that RA-related dysregulation in cytokines, adipokines, and matrix metalloproteinases (MMPs) contributes to early myocardial dysfunction and potentiates the impact of comorbidity development on clinical HF onset. In Specific Aim 1, the PI will initiate a new, prospective RA cohort with comprehensive cardiac risk stratification, biospecimen collection, and deep cardiac phenotyping using speckle-tracking trans-thoracic echocardiogram and paired cardiac magnetic resonance imaging to investigate RA-related biomolecular alterations underlying subclinical myocardial dysfunction. The hypothesis in Aim 1 is that disproportionate increases in circulating interleukin (IL)-1, IL-6, adiponectin, and MMPs in RA will associate with subclinical myocardial dysfunction as indicated by worsened systolic and diastolic dysfunction, valvular heart disease, and myocardial infiltration. In Specific Aim 2, the mediating effect of comorbidity development on clinical HF and HF subtypes will be quantified through causal mediation analyses in a national cohort of RA patients and matched non-RA controls. Further, the independent and combined effects of comorbidity with RA-related risk factors on HF risk will be examined in an ongoing, multicenter, prospective cohort of >3,500 RA patients. The hypotheses in Aim 2 are that 1) obesity, diabetes, hypertension, chronic lung disease, and valvular heart disease will mediate excess HF risk in RA, and 2) RA-specific factors interact with these comorbidities to potentiate HF risk in patients with RA. Impact: Successful completion of this study will identify high-priority, modifiable risk-factors for myocardial dysfunction in RA and inform the development of personalized HF prevention and management strategies in RA. Moreover, this mentored research will develop the PI into an independent, patient-oriented investigator focused on improving CVD outcomes in RA.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY. Chronic wounds (CWs) are currently affecting 1 – 2% of the population in the U.S. They have a devastating impact on patients’ health, functional status, and overall quality-of-life. CWs are characterized by delayed skin tissue regeneration processes due to the impaired cell migration of dermal fibroblasts, vascular endothelial cells, and keratinocytes in CWs. Current clinical standard-of-care does not specifically correct these physiological impairment. More advanced treatments, such as growth factors, microRNA-targeting therapies, and stem cells have shown a better efficacy in enhancing cell migration and skin tissue regeneration. However, they face challenges of low efficacy in human patients, inefficient delivery and low stability of therapeutics, and high cost. Due to the limitations of the current CW therapies, there is a critical need for novel treatment technologies that can be easily applied to CWs and efficaciously enhance cell migration and skin tissue regeneration without imposing a high financial burden on patients. Direct current (DC) electric field (EF) is capable of guiding directional migration and increasing the migration speed of keratinocytes, dermal fibroblasts, and vascular endothelial cells in vitro, a phenomenon called electrotaxis. Electrotaxis has also led to accelerated wound closure in in vitro scratch wound healing assays. Compared to the current technologies, electrotaxis provides unique advantages including highly directional cue for guiding cell migration, rapid cellular responses, easy treatment application, programmable treatment parameters, and low cost. Despite the successful application of electrotaxis in in vitro, the in vivo wound healing efficacy of electrotaxis is limited and insignificant. The reason for this low efficacy is that the DC EF strength that is effective in in vitro cannot be safely applied to in vivo wounds using the conventional electrical stimulation (EStim) electrodes without inducing tissue damage. Our long-term goal is to develop safe and efficacious EStim-based technologies for the CW treatment. Toward this goal, our lab recently developed a hydrogel ionic circuit (HIC) electrode that can safely apply high-strength DC EF to biological tissues by minimizing electrochemical reaction-induced interfacial pH and temperature changes. Our main objective in this proposal is to develop a novel wearable HIC-based electrotaxis system with a high flexibility and a reduced size to facilitate the healing of CWs. Our central hypothesis is that our wearable HIC-based electrotaxis system can safely apply high-strength DC EFs to promote directional migration of keratinocytes, dermal fibroblasts, and vascular endothelial cells into CW at increased speed, which will lead to accelerated CW healing. Our Specific Aims are: 1) to develop a wearable HIC-based electrotaxis system that can safely apply high-strength DC EF to induce electrotaxis cell migration; 2) to determine the in vivo safety and efficacy of our wearable HIC electrotaxis system in enhancing CW healing. If successful, our high-impact technology has the potential to enhance the clinical treatment efficacy of CWs, reduce the amputation and mortality rate, reduce the financial burden on patients, and improve the overall quality-of-life of CW patients.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Human papillomavirus (HPV) infections are responsible for almost all cases of cervical cancer, as well as most anogenital and oropharyngeal cancers. HPV, a non-enveloped DNA virus, is transported from the cell surface to the nucleus for genome replication. Recent studies have shown that, rather than exiting directly from the endosome into the cytosol after endocytosis, incoming HPV remains within a vesicular compartment and moves along microtubules (MTs) toward the nucleus. Previous research demonstrated that the C-terminus of the HPV L2 minor capsid protein contains a novel cell-penetrating peptide. This peptide facilitates L2 protrusion through the endosomal membrane, enabling its interaction with the endosomal sorting complex, retromer, which directs the virus into a retrograde transport vesicle en route to the Golgi. These processes promote the exposure of L2 protein to the cytosol, thereby facilitating the recruitment of host factors necessary for HPV retrograde transport. These findings raise important questions about how and when, after endocytosis, the HPV-containing endosomal compartment interacts with the dynein transport machinery for intracellular movement along MTs across the host cytoplasm and reach the nucleus. The discovery of dynein adaptors involved in retrograde transport highlights their roles in the precise recruitment and interaction of dynein with specific membranous compartments. This has led us to investigate the mechanisms by which dynein is recruited to HPV-carrying endosomal vesicles to mediate viral transport after endocytosis. Our recent research has uncovered a novel molecular basis for dynein recruitment to the HPV-carrying endosome. In this process, HPV utilizes the early endosomal small GTPase Rab5 and its effector Rabankyrin-5 to form a complex that links the HPV-carrying endosome to dynein, facilitating viral transport along MTs. We propose that HPV repurposes Rabankyrin-5 as a new dynein adaptor, enabling precise dynein recruitment and physical interactions with HPV-carrying endosomes. This process is linked to the exposure of HPV L2 protein to the cytosol, which facilitates Rabankyrin-5 recruitment. In Aim 1, we will determine how HPV infection triggers Rabankyrin-5-mediated virus-dynein association at the endosome, specifically investigating whether L2 protrusion is required for Rabankyrin-5 recruitment and examining the mechanism of L2-Rabankyrin-5 interaction. Using live-cell imaging, we will examine the timing of dynein recruitment relative to L2 protrusion during HPV movement along MTs. In Aim 2, we will investigate whether Rabankyrin-5 acts as a novel dynein adaptor, directly interacting with dynein and activating its function to promote HPV transport. This research has the potential to uncover a novel mechanism for HPV intracellular transport during the early stages of viral entry, involving the spatial and temporal coordination of viral membrane protrusion, endosomal coat complex formation, and precise dynein recruitment and activation. By advancing our understanding of the cell biology and molecular virology of HPV infections, this research offers molecular insights that could lead to the identification of new therapeutic targets and the development of innovative antiviral approaches.

NIH Research Projects · FY 2025 · 2025-08

The Temporomandibular Joint (TMJ) is the most-used joint in the body. The TMJ cartilage carries out the essential function of enabling free joint movement during speech and mastication. TMJ Osteoarthritis (TMJ-OA) consist of osteochondral tissue degeneration and is a growing epidemic that afflicts men and women not only in United States but across the globe. It is well established that altered expression and activation of catabolic enzymes underlies joint cartilage destruction observed in TMJ-OA, however the precise mandibular chondrocyte behavior during TMJ cartilage degeneration and regeneration is not well understood. Notch signaling has been identified as a potential catabolic and anabolic mediator of TMJ-OA. Understanding Notch cellular signaling that regulate osteochondral tissue degeneration can lead us to development of new therapeutic targets. To sufficiently advance our understanding of Notch signaling in TMJ degeneration-regeneration and translate our findings, will require robust experimentation, including preclinical studies in TMJ degeneration model. Our long-term goal is to develop a clinically relevant approach to modulate Notch signaling to prevent TMJ degeneration and facilitate its regeneration. Our central hypothesis is that Notch ligand (Jagged 1 / 2) is a potential target for TMJ- OA, and they work via upregulation of bone morphogenetic protein 2 (BMP2) and Indian hedgehog (Ihh) signaling pathway. This work will be completed in three specific aims, using novel mouse model and state of art genomic technology. Our preliminary work has demonstrated that Jagged 1 is significantly over expressed (both mRNA & protein) in TMJ degeneration both in mice and humans. In specific aim 1, we will disrupt Jagged1/2 in lineage specific manner in chondrocytes and osteoblasts of subchondral bone. In specific aim 2, we will define the pathophysiological mechanism by which Notch ligand (Jagged 1 / 2) leads to TMJ degeneration and regeneration at single cell level and in specific aim 3, our experiments will translate our mechanistic observation and study the role Jagged1/2 plays in traumatic TMJ injury and repair, specifically using anti-Jagged 1 antibodies to reduce TMJ degeneration. The proposed project has immense potential to reveal new regulatory pathways that modulates TMJ degeneration and regeneration and to open new insights on understanding the disease mechanism and developing therapeutic interventions.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT The overarching goal of my research is to improve prevention, early detection, and outcomes of infection- related cancers, particularly among people living with HIV (PHIV) who experience an increased burden. Due to the immunosuppression and aberrant immune activation that accompanies lifelong HIV infection, PHIV have a higher prevalence of oncogenic infections (i.e. HPV) and those infections are more likely to persist and progress to cancer (i.e. cervical, anal, and oropharyngeal). My hypothesis is that systemic immune dysregulation is associated with an inability to control oncogenic infections and exacerbated in PHIV. Under this Catalyst Award, I plan to grow my research in the distinct direction of understanding the immune drivers of persistent oncogenic infections. To address this, I propose to start by utilizing a novel application of genome- wide DNA methylation. This recently published method can determine a person’s immune cell profile comprised of 12 immune cell subsets from the methylation array data, which can provide information on their overall immune function, which can then be investigated for its association with persistent oncogenic infections and progression to cancer. The first project I propose towards this goal will utilize specimens already available or currently being collected to investigate the immune drivers of oral HPV persistence, the pre-cursor to oropharyngeal cancer (OPC) by assessing the association between host immune cell profile and persistent oral hrHPV. Oropharyngeal cancer (OPC) caused by oral human papillomavirus (HPV) infection is increasing in incidence globally with a burden of disease disproportionately distributed among men and PHIV. A persistent oral high-risk HPV (hrHPV) infection has been identified as the obligate precursor to HPV-associated OPC (HPV-OPC) and can be utilized as a study endpoint. Therefore in this first study, methylation-based immune profile will be compared by oral HPV persistence status to understand the degree of immune function among study participants and its association with oral hrHPV persistence. I will then plan to repeat these methods for investigation at other HPV-related anatomic sites among the same participants and in other cohorts I serve as investigator. With a research background in basic science and molecular epidemiology I will be able to integrate multiple sources of data using a novel epigenetic tool not previously used to investigate persistent oncogenic infections among PHIV. My prior work has pursued overarching research interests within HPV- associated cancer or cancer among PHIV. The new direction of my research has a foundation in my prior work and expertise but represents movement in a new direction with a focus in immuno-oncology to gain insight into the immune drivers of persistent oncogenic infection that progresses to cancer among PHIV.