University Of Nebraska Medical Center

NIH Research Projects · FY 2025 · 2024-06

Project Summary: Characterizing the Function of Cyclic di-AMP on Chlamydial Growth and Differentiation Chlamydia is an obligate intracellular bacterial pathogen that causes a range of serious diseases in humans. In developed countries, Chlamydia trachomatis is the primary cause of bacterial sexually transmitted infections (STI). Indeed, recent reports from the Centers for Disease Control highlight the increasing incidence of STIs, with chlamydia infections consistently outpacing all other bacterial causes. In developing countries, C. trachomatis is not only a significant cause of STI, but it is also responsible for the primary cause of infectious preventable blindness, trachoma. The major concern of chlamydial infections is that they are often asymptomatic and undiagnosed, which can lead to chronic sequelae. These include pelvic inflammatory disease, tubal factor infertility, and reactive arthritis for C. trachomatis. Consequently, chlamydial diseases remain a significant burden on health care systems around the world. In adapting to obligate intracellular growth, Chlamydia has significantly reduced its genome size and eliminated genes from various pathways as it relies on the host cell for its metabolic needs. This suggests that the presence of any particular gene(s) is critical, if not essential, for its growth and pathogenesis. Importantly, Chlamydia also alternates between different functional and morphological forms during its normal growth, also referred to as its developmental cycle. These observations, combined with its obligate intracellular dependence, makes Chlamydia a difficult organism with which to work. However, recent development of genetic tools to mechanistically study chlamydiae have significantly enhanced our understanding of this pathogen. Related to this proposal is the observation that Chlamydia produces a second messenger compound, cyclic di-AMP (c-di-AMP), that is known to activate host cell immune-signaling pathways. Interestingly, c-di-AMP is typically produced in Gram-positive bacteria and mycobacteria to regulate aspects of their physiology and pathogenesis, yet Chlamydia is a Gram-negative bacterium. We propose to investigate why Chlamydia produces c-di-AMP and how it functions in the physiology of the organism. The first project goal is to characterize using genetic techniques the effects of altering c-di-AMP production on Chlamydia growth and developmental cycle progression. The second project goal is to identify genes that are regulated by c-di-AMP. Results from the proposed studies will advance our understanding of the function of c-di-AMP in chlamydial physiology and identify new targets for development of Chlamydia-specific treatments.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract As our population ages, Alzheimer’s Disease (AD) and related pathologies will continue to increase in prevalence, making the investigation of modifiable and non-modifiable risk factors related to AD critical. Non- modifiable risk factors, such as genetic risk that individuals are born with, and modifiable risk factors, such as physical activity (PA) and cardiorespiratory fitness (CRF) levels, significantly influence brain development and disease pathology. Utilizing a lifespan perspective to investigate the ways in which these risk factors effect brain development during childhood, a critical period of neurodevelopment, may lead to novel insights on mechanisms and prevention of AD in the brain. This proposal aims to study the relationships between cognition, physical activity (PA), cardiorespiratory fitness (CRF), genetics, and the development AD-vulnerable brain networks in a cohort of periadolescents (aged 8-13 years old). Evidence suggests that PA and CRF can enhance the brain’s functional connections in networks most vulnerable to AD pathology, including the Default Mode Network (DMN), and the Frontoparietal Network (FPN). Investigating these networks in relation to PA and CRF during this critical neurodevelopmental period will aid in the understanding of how modifiable risk factors influence AD-vulnerable brain networks. While it is well known that APOE-ε4 status influences AD risk, more recent evidence suggests PA and CRF may help mitigate risk associated with genetic profile, however this relationship has not been investigated in developing cohorts. In order to fill the gap in this research area, cognitive, brain imaging, and genetic data will be collected as part of larger (N = 270) cohort of periadolescents enrolled in the parent study (R01 AG064247), in addition to PA monitoring and CRF testing completed as part of an additional sub-study designed by the applicant. We hypothesize that greater PA and CRF will be associated with enhanced memory and executive function ability, as well as greater within-network functional connectivity profiles that support cognition. Interactions of these findings will also be investigated in relation to APOE allele status, as we also hypothesize that PA and CRF levels may moderate the relationship between cognitive development and genetic risk. The applicant, an MD/PhD student at the University of Nebraska Medical Center, will be able to develop skills related to hypothesis generation and testing, human-subjects research methods, data analysis, and development of scholarly products. Throughout the proposed research and clinical training, advanced courses, clinical preceptorships, workshops, and hands-on experiences will be completed in order to develop the scientific skills necessary to fulfill the aims and become a rigorous and well- rounded physician scientist.

NIH Research Projects · FY 2026 · 2024-05

Project Summary/Abstract Exposure to alcohol during pregnancy produces fetal alcohol spectrum disorders (FASD) that are associated with sensory and cognitive deficits. Individuals with FASD have impaired auditory processing and frequently exhibit atypical auditory behaviors. Developmental alcohol exposure has been shown to cause impairments in neuronal plasticity and GABAergic interneuron dysfunction. Parvalbumin expressing GABAergic interneurons are surrounded by perineuronal nets that regulate their development and plasticity. However, it is not known whether altered development of perineuronal nets and maturation of parvalbumin interneurons contribute to altered inhibition and changes in the network excitation in the auditory system in mice prenatally exposed to alcohol resulting in impairments in auditory processing. Here, we will use a mouse model of maternal voluntary alcohol consumption throughout gestation to examine the functionality of the central auditory pathway, identify region specific deficits in perineuronal nets and parvalbumin interneurons in the auditory system, examine synaptic inhibition in the primary auditory cortex and inferior colliculus, and investigate hyperexcitability in the primary auditory cortex in vivo. The goal of the proposal is to understand the effects of prenatal alcohol exposure sustained across gestation on auditory processing and identify auditory structures and cellular correlates that contribute to the auditory impairments.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Background: Dolutegravir (DTG) is a first-line antiretroviral drug used in combination therapy for the treatment of human immunodeficiency virus type-1 (HIV-1) infection. Due to the roll out of generic DTG-based regimen, its inclusion in national treatment guidelines and rising pretreatment resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in resource limited countries, in just 5 years, 15 million HIV-1 infected people will be treated with DTG. This includes women of child-bearing age, who remain a significant infected population (UNAIDS data, 2021). However, during recent years, growing data from clinical and pre-clinical studies have suggested that DTG is associated with developmental neurologic abnormalities. Thus, concerns emerged for the usage of DTG-based regimens in pregnant women or those of child-bearing age. Knowledge gap: Underlying mechanism for DTG-associated developmental neurotoxicity (prenatal and postnatal), particularly in babies born without structural, brain or spinal cord, malformations, remains unknown. Moreover, therapeutic measures to enhance the DTG use for safer medication during pregnancy are infancy. Our preliminary data: DTG was found to be a broad-spectrum inhibitor of MMPs. The drug was found to bind Zn++ at the catalytic domain, leading to inhibition of MMPs activities. Moreover, studies in pregnant mice showed that DTG can cross the placental barrier, accumulate in the fetal CNS and inhibit MMPs activity during the critical period of brain development. Further postnatal evaluation of brain health in mice pups following in utero DTG exposures identified neuroinflammation, neuronal damage and behavioral deficits. These data demonstrated an association between DTG dysregulation of MMPs activities during gestation and consequent neurotoxicity. Hypothesis: We posit that DTG inhibition of MMPs activities impairs pre- and post-natal neurodevelopment and offered long- acting approaches will serve to improve drug’s therapeutic benefits during pregnancy. Research Strategy: (1) Determine longitudinal dose-dependent effect of DTG on MMPs activity and/or expression in brain of embryos and early age postnatal pups and identify impact of such inhibitions in MMPs activity on neuronal development. (2) Investigate postnatal neurobehavioral outcomes that occurs across mouse development from adolescence to young age following gestational DTG exposure. (3) Utilize long-acting poloxamer-based DTG nanoformulations to provide potential delivery means to attenuate drug-associated neurotoxicity. (4) Establish scientific exchange with clinical study to establish clinical relevance. Outcome: Successful outcome of the proposal will significantly advance the basic and clinical research fields in understanding the theory of DTG inhibition of MMPs activities as the underlying mechanism for DTG-associated developmental neurotoxicity, impaired neurobehavioral outcomes, and provide long-acting poloxamer-encapsulated DTG nanoformulations as potential delivery means to improve the safety and therapeutic profile of DTG during pregnancy.

NIH Research Projects · FY 2026 · 2024-05

The goal of this research is to characterize Ras-driven and KSR1-dependent mechanisms that modulate tumor initiation, drug resistance and the epithelial-to-mesenchymal transition (EMT) in colorectal cancer. Tumor initiating cells (TICs) are a subpopulation of tumor cells defined by their ability to self-renew and regenerate the entire heterogeneous tumor population and are believed to be a reservoir of drug resistant persister cells. EMT- like behavior contributes to tumor initiation, invasion, and metastasis. Partial reversal of EMT to a “hybrid” state is necessary for efficient TIC formation and metastatic colonization. In turn, TICs lose clonogenicity and self- renewal and acquire the invasive behavior of mesenchymal cells that drives their dissemination. While the interconversion of tumor cells to migratory and invasive mesenchymal cells from self-renewing TICs is necessary for metastasis, little is known about the mechanisms that direct tumor cells between these phenotypes. We combined targeting of genes encoding key effectors of Ras signaling with polysome profiling and computational analysis to discover that the molecular scaffold KSR1 mediates resistance to clinically approved drugs and regulates TIC formation and EMT via RNA binding protein SRSF9. Our preliminary data suggest the hypothesis that KSR1- dependent signaling in KRAS-mutated CRC promotes the splicing and translation of mRNAs critical to CRC tumor initiation, drug resistance, and EMT. Using genetically modified CRC organoids, mouse models, and cells, the synergistic expertise of our team will characterize these novel pathways controlling CRC behavior in vitro and in vivo by 1) Characterizing the KSR1-dependent pathway(s) necessary for tumor initiation and resistance, 2) Defining the KSR1-dependent mechanism(s) driving cells from partial EMT toward invasive mesenchymal behavior, and 3) Determining how KSR1-dependent splicing promotes translation of proteins critical for EMT. Completion of this research will reveal novel mechanisms that may be targeted to improve therapeutic response and inhibit tumor initiation and metastasis.

NIH Research Projects · FY 2025 · 2024-04

Abstract: Although there is an 8% decrease in HIV infection in the U.S., the prevalence of people with HIV (PWH) has increased due to effective combinational antiretroviral therapy (cART). PWH are prone to substance abuse such as methamphetamine (METH), opioids, cannabis, and alcohol. Moreover, HIV and substance abuse constitutes a health syndemic and contribute to a significant economic burden to the U.S. PWH and, with METH abuse, have activated and inflamed immune systems, increasing the risk of neurocognitive disorders (NCD). HIV and METH increase neuroinflammation, whereas cannabidiol (CBD), a component of cannabis, is known to attenuate inflammation; however, their collective impact is yet to be elucidated. Thus, there is a critical need to delineate the mitigating effect of CBD on neuroinflammation in HIV infection and METH abuse. Our long-term goal is to establish effective intervention strategies to enhance PWH's span and quality of life. Our overall objective for this proposal is to understand the impact of CBD on HIV infection and METH abuse- associated neuroinflammation. In recent times, extracellular vesicles (EVs) have gained considerable attention as novel actors in intercellular communication, inflammation, and disease progression. On the other hand, depending on the cell of origin, EVs can have precisely the opposite effect, i.e., alleviate inflammation. Several preclinical studies have indicated CBD alleviates inflammation by inhibiting NLRP3 inflammasome activation and cytokine production. However, the underpinning mechanism of CBD's effects on neuroinflammation in the context of HIV infection and METH abuse has not been explored. Based on the previous findings, we hypothesize that CBD attenuates HIV and METH abuse-associated neuroinflammation by modulating NLRP3 inflammasome activation and altering the EV cargo. We will test this hypothesis under the following two aims; In Aim 1, we will evaluate the effect of CBD in modulating NLRP3 inflammasome activation and EV cargo in vitro. We will use human monocyte-derived microglia (MDMi) in our study. RNA and protein isolated from different experimental groups will be used to analyze gene expression, whereas EVs isolated from conditioned media will be subjected to cytokines analysis. In Aim 2, we will investigate the effect of CBD on neuroinflammation and EV cargo in HIV infection and METH abuse using a Hu-mice model. Preclinical hu-mice models with METH administration and HIV infection that exhibit pathophysiological complexities of the human brain will accurately mimic neuroinflammation in clinical settings. Thus, delineating the impact of CBD on the modulation of neuroinflammation in the context of HIV infection and METH abuse will identify genes involved in neuroinflammation and NLRP3 inflammasome activation during HIV infection and METH abuse and the impact of CBD administration.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Survivors of critical illness frequently experience profound physical, mental, and cognitive health impairments that are initiated and/or exacerbated by known racial and socioeconomic health disparities and outdated intensive care unit (ICU) mechanical ventilation and symptom management practices. This morbidity is potentially preventable through the application of the ABCDEF bundle; a multicomponent, evidence-based intervention to improve team-based care. While consistently proven safe and effective, national ABCDEF bundle performance remains unacceptably low as clinicians continue to struggle with multiple barriers to bundle delivery. The long-term goal of the proposed work is to develop pragmatic and sustainable strategies to increase the delivery of evidence-based practices that lead to improved care for critically ill adults across a variety of healthcare systems, particularly those serving populations with known health disparities (safety net hospitals). Our overall objective is to evaluate two strategies grounded in behavioral economic theory and implementation science to increase ABCDEF bundle adoption. The strategies being evaluated target a variety of ICU team members and known behavioral determinants of bundle performance. The proposed project includes two phases and four aims. In Phase 1 (UG3), we will work with the NIH’s Healthcare System Research Collaboratory Coordinating Center and our community partners to meet key milestones aimed at enhancing and finalizing the implementation strategies and research methods used to facilitate and evaluate the effectiveness of ABCDEF bundle adoption (UG3 Aim 1). In Phase 2 (UH3), we will conduct a pragmatic, stepped-wedge, cluster randomized hybrid type III effectiveness-implementation trial. After creating 6 matched pairs of 12 ICUs from 3 hospitals (N=8,100 patients on mechanical ventilation), we will randomly assign ICUs within each matched pair to receive either real-time audit and feedback (strategy A) or a Registered Nurse (RN) implementation facilitator (strategy B) and each pair to one of six wedges. The aims of the trial are to compare the effectiveness of real-time audit and feedback and RN implementation facilitator on ABCDEF bundle adoption (UH3 Aim 1; primary outcome) and clinical outcomes (i.e., duration of mechanical ventilation; ICU, hospital, and 30-day mortality; ICU and hospital length of stay; days with acute brain dysfunction; discharge disposition, psychoactive medication, and physical therapy utilization; and 30- day hospital readmission) (UH3 Aim 2). Finally, we will identify and describe key stakeholders’ experiences with, and perspectives on, the acceptability and impact on workload of the implementation strategies (UH3 Aim 3). We expect study results will impact the field by developing simple, yet effective, ways of accelerating the reliable uptake of a variety of evidence-based ICU interventions that will address known health disparities in the ICU and ultimately improve the care and outcomes of millions of critically ill adults annually.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal, treatment-refractory malignancy characterized by fibroblast-rich stroma. While external beam radiation therapy (EBRT) is a staple in the current treatment of PDAC, its therapeutic efficacy is limited by normal tissue toxicities and its inability to target metastatic disease. Targeted radionuclide therapeutics (TRTs), particularly low-molecular-weight carriers, offer an avenue to improve the deliverable radiation dose and therapeutic efficacy compared to EBRT by selectively targeting tumors (both primary and metastatic) and substantially reduce non-target toxicities. The fibroblast activation protein-α (FAP) is selectively expressed on tumor-associated fibroblasts in the tumor microenvironment (TME) of PDAC (~75%) and other malignancies. Several clinical imaging studies have validated the potential of small molecule, FAP- targeted constructs (e.g., 68Ga-FAPI-46). However, the therapeutic translation of FAP-TRTs has failed mainly due to their low tumor-residualization, which diminishes deliverable therapeutic doses, therapeutic efficacy, and clinical potential. Recently, we have developed an endolysosomal trapping approach reliant on adduct formation with cysteine protease to increase tumor residualization of TRTs substantially. Using this adduct formation approach, the goals of this proposal are to optimize 177Lu- and 225Ac-FAP-targeted TRT constructs, obtain a greater understanding of how PDAC biology impacts adduct formation, demonstrate our construct's therapeutic efficacy and safety in PDAC models, understand the impact of FAP-TRTs on the PDAC tumor microenvironment and compare and contrast the biological effect of 177Lu (β-) vs. 225Ac (α) based FAP-TRTs. Another challenge for FAP-targeted therapies emerges from its heterogeneous expression and the inadequacy of preclinical models to recapitulate variabilities in FAP expression. We have generated a panel of human immortalized patient-derived pancreatic fibroblasts (iPDPFs) that recapitulate CAF heterogeneity and variable FAP expression. We have also developed unique CAF-tumor cell co-implantation models that result in FAP high or FAP low tumors. We hypothesize that incorporating cysteine protease trapping agents (CPTAs) into the structure of FAP-targeted agents (FAPTAs) will allow the formation of intracellular adducts capable of delivering higher therapeutic efficacy through enhanced PDAC-specific retention. To test this hypothesis, two specific aims are proposed. Aim 1 focuses on optimizing 177Lu/225Ac-CPTA-FAPTAs and examining adduct formation and distribution in PDAC models. Studies in Aim 2 are designed to evaluate the therapeutic efficacy of 177Lu/225Ac-CPTA-FAPTAs and investigate the impact of FAP-TRT therapy on CAF heterogeneity. We propose to employ cutting-edge approaches and models to study the biodistribution, cellular uptake and trafficking, adduct formation, dosimetry, and therapeutic efficacy of 177Lu/225Ac-CPTA-FAPTAs. Successful completion of the proposed studies will provide the critical data required to progress the novel FAP-TRTs toward regulatory approval and initiation of clinical trials in the future.

NIH Research Projects · FY 2026 · 2024-03

Project Summary Electronic Nicotine Delivery System (ENDS) use has been a major public health concern. The U.S. Food and Drug Administration (FDA) continues to cite health effects as a chief rationale in FDA enforcement priorities and marketing denials. Childhood to adolescence represents a critical period with rapid development in brain maturation. Combustible cigarette smoking during adolescence can negatively impact brain development, and animal models also show that nicotine exposure in adolescent rodents slows brain maturation, including the prefrontal cortex. However, the impact of rising ENDS use on youth brain functions and cognitive outcomes is poorly understood. Vaping delivers much lower levels of tobacco smoke chemicals but similar levels of nicotine compared to cigarette smoking. Studying the effects of vaping on brain maturation can illuminate nicotine effects without tobacco smoking. Importantly, there is a dearth of evidence on the relative effects of exclusive ENDS use vs. combustible cigarette smoking on youth brain maturation, which is critical to the FDA’s regulatory actions on ENDS and prioritizing public health programs. This population study will analyze the longitudinal Adolescent Brain Cognitive Development (ABCD) study with 11,878 youth aged 9-10 years old at baseline from 21 U.S. study sites, to assess the effects of exclusive ENDS use on youth’s brain structure and cognitive performances and further examine potentially differential effects of vaping vs. smoking on neurocognition. The study will link the first 5 biannual waves of ABCD magnetic resonance imaging (MRI) data during 2016-2024 with semiannual tobacco use assessments and annual behavioral measures by Subject ID to create a harmonized longitudinal dataset. In Aim 1, the team will prospectively examine the effects of exclusive ENDS use (vs. never tobacco use) on whole-brain morphometric measures (e.g., white/gray matter, cortical thickness). In Aim 2, the team will assess the longitudinal associations of exclusive ENDS use (vs. never tobacco use) on attention, memory, executive function, emotion regulation, and reward processing at the cognitive level, using validated behavioral tasks sensitive to youth development (e.g., NIH Toolbox), and at the brain level using functional MRI tasks. In both Aims 1 and 2, we will further test whether differences in brain maturation are negatively associated with vaping frequency, intensity, and duration among ENDS users. In Aim 3, the relative effect of exclusive ENDS use vs. cigarette smoking on whole brain structure and NIH Cognition Toolbox will be tested. This study is innovative by 1) leveraging the largest brain development and child health study with state-of-the-art neurocognitive assessments, and 2) applying the mariginal structural modeling to handle time-dependent exposure and time-varying confounders, thus allowing for unbiased effect estimation and teasing out potentially reverse causality. This study is also significant in providing vital evidence on vital evidence of vaping effects on brain health, thus providing vital evidence to inform effective regulatory actions and public policies. 1

NIH Research Projects · FY 2025 · 2024-03

Project summary: Despite combined antiretroviral therapy, HIV proteins like Transactivator of Transcription (Tat) persist at low levels in the brains of HIV-infected individuals, resulting in cognitive decline collectively referred to as HIV-1- associated neurocognitive disorders (HAND). Alcohol use disorder is a common comorbidity in people living with HIV (PLWH) and is also known to potentiate the pathogenesis of HAND. Although the mechanisms underlying HAND are complex and remain poorly understood, low-level, persistent neuroinflammation has been shown to be a correlate of HAND. In the brain, glial cells such as the astrocytes, are major contributors of inflammation, involving the inflammasome NLRP6 (a member of the NLR [nucleotide-oligomerization domain-like receptor) family of proteins that acts as a sensor protein and plays multiple roles in regulating inflammation and host defenses. Based on the premise that alcohol use exacerbates HIV-associated neuroinflammation, we hypothesize that exposure of HIV Tat stimulated astrocytes to alcohol will lead to exacerbated expression of a pro-inflammatory milieu compared to cells exposed to either agent alone or to control cells (HIV protein/drug naïve) and, further that mechanistically this involves increased activation of NLRP6 and its downstream signaling pathways. In fact, our exciting preliminary studies have demonstrated that human A172 astrocyte cells exposed to both HIV Tat (surrogate of HIV infection) and alcohol significantly increased the activation of NLRP6 involving endoplasmic reticulum (ER) stress. The proposed hypothesis will be tested in two aims: SA1 To investigate the molecular mechanism(s) underlying HIV Tat and ethanol-induced astrocytic activation in mouse primary astrocytes (MPAs) and SA2. To validate the findings from SA1 in an in vivo doxycycline-inducible transgenic Tat (iTat) mice model exposed to ethanol These findings could have important implications for the future development of therapeutic interventions aimed at mitigating neuroinflammation in PLWH with HAND and alcohol use disorder.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Mutations in the human genome give rise to many human diseases including cancer. Mutations are also beneficial for processes including evolution and adaptive immunity. However, little is known about the molecular basis of mutagenesis. One mechanism that leads to the introduction of mutations is misincorporation of nucleotides by DNA polymerases during DNA replication and repair. The focus of our application is on deoxynucleoside triphosphate (dNTP) substrate selection by DNA polymerase beta (Pol β). Pol β functions in base excision repair, a process that has evolved to repair oxidative DNA damage. These types of damage are generated by a cell’s endogenous metabolism as well as exogenous sources such as ionizing radiation, UV light, and chemotherapeutic agents. During base excision repair, 20,000-50,000 predominantly oxidative DNA lesions are enzymatically removed, leaving small gaps in the DNA that are filled in by Pol β. Incorrect substrate selection by Pol β during base excision repair has potential to result in genomic instability. We have recently shown that Pol β also functions in DNA gap filling during microhomology-mediated end-joining in VDJ recombination, where it plays an important role in generating immune diversity. Our broad, long-term objective is to understand how DNA polymerases choose the correct substrate for incorporation into DNA. Pol β is an excellent model for studying polymerase mechanisms because of its relatively small size and the ease of purifying protein and growing crystals. The aims of this application are to understand how Pol β selects the correct dNTP and why it sometimes chooses the incorrect dNTP for incorporation into DNA. We will employ a powerful combination of biochemical, structural, biophysical, computational and biological approaches in this multi-investigator project. We will determine how Pol β selects the correct substrate for incorporation into DNA and how the mechanisms differ for mutator variants of Pol β. Our research will result in the characterization of the roles of amino acid residues in the selection of substrates during Pol β catalysis. Our findings will be of fundamental importance in understanding the molecular basis of substrate selection by Pol β that will likely have broad applicability to other DNA polymerases.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY: Endothelial-to-mesenchymal transition (EndMT) has been detected in atherosclerosis, and the extent of EndMT observed in the human plaques strongly correlates with the severity of the disease, implying clinical relevance of EndMT in the pathogenesis of atherosclerosis. Both disturbed blood flow (d-flow) and interleukin-1 signaling (IL-1) have been implicated in EndMT formation. The signaling pathway that links d-flow and EndMT is still unclear. Moreover, IL-1 as a therapeutic target has already been tested and the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) assessed the therapeutic effect of the anti-IL-1β antibody in patients after myocardial infarction and showed 31% reduction of cardiovascular and all-cause mortality. However, some of those patients developed immunosuppressive off-target effects. In addition, there is no clear principal cell type in human severe atherosclerosis responsible for IL-1 signaling. Our recently published work has demonstrated that d-flow induces activation of interleukin-1 receptor signaling kinase (IRAK1) within endothelial cells (ECs), with an increase in p-IRAK1 levels in human and mouse atherosclerosis. IRAK1 is co-expressed with EndMT in severe atherosclerosis, and deletion of IRAK1 decreases EndMT in vitro, identifying a novel role for IRAK1 as a downstream effector in d-flow-induced EndMT. Our central hypothesis is that IRAK1 activation mediates d- flow-induced EndMT and selective inhibition of IRAK1 will allow efficient inhibition of atherosclerosis without promoting significant immunosuppression. Aim 1 Determine the mechanistic role of IRAK1 activation in oscillatory flow-induced EndMT. Our preliminary data supports IRAK1 activation by d-flow and that mediates EndMT formation. Aim 1.1, utilizing CRISPR Cas-9 KO selective deletion and re-expressing vectors, we will test the role of IRAK1 in oscillatory flow-induced EndMT. In Aim 1.2, we will evaluate the activating roles of IRAK1 domains by transiently transfecting IRAK1 truncated mutants into IRAK1-depleted and overexpressed ECs and we will measure Phospho-IRAK1 and EndMT after oscillatory flow. In Aim 1.3, we will assess the functional role of IRAK1 endogenous interactions by utilizing immunoprecipitations, gene silencing, and mass spectrometry. Aim 2 will determine if selective inhibition of IRAK1 in d-flow-induced EndMT prevents atherosclerosis progression. We will use Cre-lox endothelial-specific lineage tracing and novel endothelial-specific IRAK1 knockout mice and IRAK1 selective inhibitor to provide the first assessment of IRAK1 inhibition on EndMT formation in vivo. Fibrous cap thickness, plaque lesion size, composition, and EndMT will be assessed in both partial carotid ligation model of d-flow (Aim2.1) and diet-induced atherosclerosis (Aim2.2). FACS analysis of blood and plaques will be done to assess total and differential leukocytes and proinflammatory cytokines. These studies will determine the dynamic interplay between disturbed flow and IL-1 signaling in EndMT and will define a novel role for IRAK1 as a novel therapeutic target in d-flow-induced EndMT and atherosclerosis.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Diabetes-induced imbalance of autonomic efferent neuronal tone (reduced parasympathetic activity and increased sympathetic activity) is involved in sudden cardiac death and is responsible for high mortality in diabetic patients. Increasing cardiovascular vagal tone significantly reduces the mortality. Although cardiovascular vagal function is severely damaged in diabetic patients, the potential mechanisms concerning reduced cardiovascular vagal function in diabetes are poorly understood. Cardiovascular postganglionic vagal neurons in intracardiac ganglia modulate the acetylcholine release via producing cell excitation and finally regulate cardiovascular function. Our previous studies have shown that nicotinic acetylcholine receptor (nAChR) currents and cell excitability are reduced in vagal neurons, which contribute to cardiovascular vagal dysfunction in type 2 diabetes mellitus (T2DM). Based on our previous studies and preliminary data, we hypothesize that leptin resistance-uncoupling protein 2 (UCP2)-hydrogen peroxide (H2O2) signaling pathway and norepinephrine- α1 adrenergic receptor-UCP2-H2O2 axis inactivate nAChR channels and further contribute to cardiovascular vagal dysfunction in T2DM. Using multi-faceted technical approaches (from whole animals to cellular-molecular levels) in sham and high-fat diet/low-dose streptozotocin-induced T2DM rats, we design in vivo and in vitro studies to verify above hypotheses. In Specific Aim 1, we will measure if H2O2 overproduction in vagal postganglionic neurons decreases nAChR currents and induces cardiovascular vagal dysfunction in T2DM. In Specific Aim 2, we will test if leptin/leptin receptors influence nAChR currents and cardiovascular vagal function in T2DM. In Specific Aim 3, we will determine if sympathetic neurotransmitter norepinephrine-α1 adrenergic receptor axis reduces nAChR currents and cardiovascular vagal function in T2DM. These studies will further our understanding of the cellular and molecular mechanisms underlying the impairment of vagal neuronal function in T2DM and will discover potential therapeutic targets for improving cardiovascular vagal function and reducing mortality in the T2DM state.

NIH Research Projects · FY 2025 · 2024-02

Advanced high-throughput technologies have transformed biomedical research into a data-intensive discipline, in which the rate of data collection and the complexity of data sets have exceeded the ability of classically trained scientists to extract and assimilate meaningful information. Today, This proposal is motivated by the need to build this critical workforce of biologists armed with cross-disciplinary training in computational, quantitative and analytical realms to harness the power of ‘Big Data’ in biomedical sciences and recognition that demand for these trained scientists represents a unique opportunity to promote diversity, equity, and inclusion in science. This training program at the University of Nebraska Medical Center (UNMC) will be administered by the Department of Genetics, Cell Biology and Anatomy, a campus leader in medical and graduate teaching that hosts key faculty serving as directors for data-rich core facilities such as next- generation sequencing, bioinformatics and systems biology, and research information technology. The proposed two-year training program derives from the natural synergy of this in-house expertise with two department sponsored pre-doctoral graduate programs in Molecular Genetics and Cell Biology (MGCB) that focuses on biological and disease mechanisms using high-throughput -omics approaches and Bioinformatics and Systems Biology (BISB), which emphasizes on novel algorithm development and computational and statistical training to advance design and analysis of big data experiments. The proposed program requests funding for three pre-doctoral traineeships in the first year and six in the subsequent years with a two-year trainee rotation. Participants will be selected concurrent with application to UNMC graduate programs and matriculating students will receive training from preceptors representing 38 laboratories in 14 basic science and clinical departments at UNMC and our sister campus, University of Nebraska Omaha. Trainees will take courses in bioinformatics, statistical analysis, biological networks, and research design and scientific thinking to provide a common skill set and language for interdisciplinary research. Workshops, seminars, and a team- based project will develop essential skills for collaborating in a ‘Big Data’ world and presenting analyses of complex data sets to diverse audiences as well as promote a sense of belonging, a key factor in achievement and retention of underrepresented minority students. The program directors are counseled by an internal advisory committee composed of faculty with experience in student mentoring and directing training programs, and a team of external advisors with experience developing T32 and other training programs. Collectively, this hierarchy of advisors and mentors will ensure that the program produces the next generation of leaders in ‘Big Data’ biomedical research, which are essential to workforce development in Nebraska and the surrounding region that include several IDeA states.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Environmental lung diseases are preventable disorders caused or made worse by adverse, inhaled exposures to inflammation-inducing aerosolized agents such as endotoxin (lipopolysaccharide/LPS) and microbial component-enriched organic dust. Every situation along the exposure continuum from long-term, low-level exposure to one-time, high-dose exposure can cause lung injury and subsequent disease. Anthropogenic climate change and associated industrial and agricultural intensification synergistically elevate worker and non-worker risk of adverse respiratory health outcomes. Environmental inhaled exposures can cause significant lung and airway inflammatory diseases including asthma, chronic bronchitis, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). Despite advances in understanding the key inflammatory signaling pathways involved in initiating the lung inflammatory response, there remains a paucity of knowledge and efficacious therapeutic options to hasten recovery and halt progression towards a chronic disease state. Our recent studies strikingly demonstrate environmental exposure-induced recruitment and activation of distinct lung monocyte- macrophage subpopulations involved in disease pathogenesis. Additionally, our bulk RNA sequencing, single cell (sc) RNA sequencing, and monocyte/macrophage subpopulation-specific transcriptomic analyses of lung homogenate harvested from mice given inhaled environmental exposures robustly implicate the aconitate decarboxylase 1 (ACOD1)-itaconate immunometabolic pathway as a potential central regulator. The proposed studies herein will be the first to investigate environmental exposure-induced lung disease through an immunometabolic lens. To this end, I hypothesize that the ACOD1-itaconate axis operates as a critical, negative regulator of lung monocyte/macrophage inflammatory processes in environmental exposure-induced lung injury and inflammation. In Specific Aim 1, I will determine the functional effect of ACOD1-itaconate pathway induction and modulation on human monocyte-derived macrophage differentiation and effector responses amidst environmental inflammatory exposures. In Specific Aim 2, the role of the ACOD1 signaling pathway in mediating lung inflammatory, resolving, and functional processes will be assessed in ACOD1 deficient mice exposed to inhaled, environmental inflammatory agents. Additionally, I will determine the therapeutic potential of lung- targeted, exogenous itaconate administration in hastening recovery following inhaled environmental exposure. The results of these studies will have an important positive impact by establishing the pre-clinical groundwork for understanding the ACOD1-itaconate axis in the context of environmental exposure-induced lung disease. Completion of these aims will optimally inform the development of novel therapies capable of preventing irreversible lung disease post-environmental exposure.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Malaria remains a global health disease that affects 40% of the world’s population and killed 619,000 in 2021. The etiologic agent of malaria are Plasmodium spp. parasites, of which Plasmodium falciparum is the most prevalent and deadly species. The World Health Organization recommends artemisinin-based combination therapies (ACTs) as first-line treatment for falciparum malaria, and dihydroartemisinin (DHA) is the active metabolite of all clinically-used artemisinins. Artemisinin resistance (ART-R) has been documented globally and is prevalent in the Greater Mekong Subregion of Southeast Asia, presenting a major hurdle to malaria eradication. The most well-characterized genetic marker of ART-R is mutations in the propeller domains of Kelch13, a protein that structurally resembles an E3 ubiquitin ligase adaptor. Our studies utilize isogenic parasites of Cambodian origin with mutations in Kelch13 and the proteasome. We have shown that P. falciparum-specific proteasome inhibitors kill ART-R parasites, and that mutations in the proteasome increase susceptibility to DHA. Moreover, proteasome inhibitors synergize with distinct classes of antimalarials that perturb proteostasis, including DHA. We and others have shown that DHA non-specifically and promiscuously alkylates heme and nearby parasite proteins. However, whether alkylated proteins are ubiquitinated and subsequently targeted to the proteasome for degradation remains unknown. In addition, it is unknown if these alkylated products are responsible for the parasite proteasome inhibition observed with DHA. Proteasome inhibition will lead to buildup of proteins that activate the unfolded protein response (UPR). In this regard, we observe that early parasite responses dictate eventual outcomes. Artemisinin-sensitive (ART-S) parasites demonstrate hyperactivation of the UPR at early ring stages and a subsequent inability to recover from UPR activation. Collectively, these data led us to hypothesize that a functional ubiquitin proteasome system (UPS) is necessary for ART-R. In Aim 1, we examine the role of the proteasome in ART-R. We will determine whether alkylated proteins are ubiquitinated and whether DHA-mediated alkylation inhibits the proteasome. In Aim 2, we examine the role of ubiquitination in ART-R. We will identify and interrogate the role of distinct ubiquitin branch patterns in conferring artemisinin survival. In Aim 3, we will determine if dysregulation of the UPS and UPR are generalities underlying artemisinin susceptibility. We will examine African parasites that are isogenic for mutations in coronin, AP2µ, and UBP1, proteins involved in endocytosis and ubiquitination that have been confirmed by gene editing to mediate ART-R, thus extending the implications of our earlier findings. An understanding of the molecular underpinnings of DHA and proteasome inhibitor synergy could extend the clinical utility of artemisinins, support proteasome inhibitor-based combination therapies, and lead to development of other antimalarials that perturb parasite proteostasis. The proposal aims to address knowledge gaps in parasite proteostasis with the intent of leveraging this knowledge for development of therapeutics to combat ART-R malaria.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Our laboratory is involved in structure-function studies of DNA replication. Over the next five years we propose to carry out two related projects, one focusing on human DNA replication and the other on replication of herpes simplex virus type 1 (HSV1) DNA. Knowledge gained from these projects about the structural basis of the fundamental processes enabling precise replication of human genome and replication of viral DNA will provide a foundation for the development of novel therapies to treat a wide variety of diseases. Project 1. High-fidelity genome replication is the foundation of healthy life. The bulk of human DNA replication is performed by the B-family DNA polymerases. Primase-polymerase α (Primosome) synthesizes chimeric RNA-DNA primers, before switching them to Polε and to Polδ for the start of leading and lagging strands replication, respectively. Despite recent progress in structural studies of B-family DNA polymerases, significant gaps remain in our knowledge regarding the mechanisms of their function. This incomplete understanding warrants additional study, especially of the determinants that tightly coordinate polymerase transactions at the replication fork. Consequently, revealing step by step the details of human DNA replication events and the coordinated action of the involved proteins remains a key project in our laboratory and is the major focus of the current project. Recently, based on the initial structural and functional characterization of Primosome, we proposed a new direction of study examining how the C-terminal domain of the primase large subunit, along with a chimeric RNA-DNA primer, fulfills the role of global regulator of transactions at the replication fork. Here, we will expand these studies using multiple approaches to characterize human Primosome transactions. Project 2. The human herpesvirus (HHV) family includes eight members grouped into α, β, and γ subfamilies, which cause a variety of diseases. The replisomes of all HHVs require six essential components: a single- strand DNA-binding protein, a two-subunit DNA polymerase complex, and a three-subunit helicase-primase (HP) complex. Approved therapies for HHV infections mainly target inhibition of the polymerase subunit, but the HP complex is an even more attractive target since its DNA unwinding and primer synthesis functions precede the processive polymerization. The search for HP inhibitors (HPIs) is ongoing, but success has been limited to α-HHVs. Improvement of current HPIs and the development of novel HPIs targeting the members of the β and γ subfamilies are complicated by the lack of knowledge about HP structures and function, as well as the detailed mechanism of inhibition. HP is an attractive target for comparative analysis with its human counterpart, since the DNA unwinding and primer formation by HP subunits occur in a significantly compact complex and requires precise coordination of both activities. Here, we propose to commence studies of HHV replication with comprehensive structure-function analysis of HSV1 HP and its mechanism of inhibition.

NIH Research Projects · FY 2026 · 2024-01

Project Title: Nebraska Center for Heart and Vascular Research Project Summary: Overall Center Organization and Management Plan Heart and vascular diseases (HVD) are the leading cause of death and hospitalization in the United States, Nebraska included. The goal of the Nebraska Center for Heart and Vascular Research (CHVR) is to build the physical and intellectual resources at the University of Nebraska Medical Center (UNMC) to propel the ability of our faculty to conduct research that drives the heart and vascular disease (HVD) field forward. Our center has exceptional institutional support, is well-aligned with UNMC’s areas of research emphasis, and we have an outstanding baseline cadre of investigators with expertise in HVD disease. The main objective of this Phase I COBRE is to develop a center that promotes and supports excellence in HVD research through the development of early career researchers, promotes collaborative research that is critical to solving complex research problems, and expands support for all HVD research with cutting edge infrastructure and mentoring activities. We have selected research project leaders with research projects that have a strong connection to the focus of the COBRE and are poised to become independent researchers. The COBRE will maximize the ability of the Center to boost HVD research at UNMC, enhance faculty research trajectories, mentor early career investigators to productive research careers, and reduce barriers to research to drive discovery. The Center has brought together the full range of cutting-edge expertise needed to accomplish our mission and assembled a strong mentoring program that will train mentors in best practices. To catalyze cutting edge HVD research the Center will support existing resources and grow new ones like the proposed Bioassay Core focused on the targeted examination of proteins. With these goals and the Phase I COBRE funding, the Center will support the infrastructure required for cutting-edge HVD research, increase the number of funded researchers, and total HVD funding to promote a reputation and culture of excellence in HVD research that can improve patient outcomes and reduce HVD health disparities.

NIH Research Projects · FY 2026 · 2023-12

Peripheral nerve injuries (PNI) affect millions of people in the US, and PNI with large gaps require surgical repair. Although biological and synthetic grafts are widely used to repair PNI with large gaps, they both can suffer from suboptimal clinical outcomes. Autografts are the gold standard treatment but are limited by availability and defect repair size, while synthetic grafts have poor biodegradability, strength, bioactivity, and functionality. Thus, the long-term objective of this proposal is to engineer grafts with enhanced large-gap nerve regeneration capabilities. Physical and chemical stimulation can enhance nerve regeneration responses, thus, incorporating these modalities into engineered grafts may address some current treatment limitations. Electrical stimulation (ES) can enhance nerve conduction, neurotrophin release, and functional recovery of nerve crush injuries, but these benefits have not been established for large-gap PNI. Chemical stimulation using 4-aminopyridine (4-AP; a potassium channel blocker) appears similar to ES in its effects on neurons and can enhance crush PNI repair, yet may act synergistically with ES. Implementing these physical and chemical cues for effective large-gap PNI repair will require surgical insertion of an electrically conductive scaffold with appropriate mechanical strength, degradation, conductivity, and pore properties. This proposal aims to deliver 4-AP and ES via novel, biodegradable, ionically conducting (IC) chitosan scaffolds and hybrid-engineered nerve allografts to repair large- gap nerve defects. We found that IC scaffolds with ES+4-AP treatment in large-gap nerve defects increased neurotrophin release, myelination, compound action potential, and gastrocnemius muscle weight beyond ES or 4-AP alone. Increased blood vessel growth and reduced fiber capsule thickness surrounding scaffolds with ES+4-AP treatment indicate improved biocompatibility and regeneration. Significantly higher levels of genes for TrkA, TrkB, and TrkC receptors and NGF, BDNF, NT3, and CD31 were found for ES+4AP treatment important for nerve regeneration. Therefore, it was hypothesized that IC scaffolds combined with chemical and electrical cues will modulate cell-material interactions to enhance axon regeneration rate and functional recovery comparable to autografts. This will be tested with three Specific Aims: 1) Characterize ionically conducting (IC) scaffolds with variations in drug release rate, conductivity, and biodegradation; 2) Assess human and rat Schwann cell responses to IC scaffolds with 4-AP and/or ES in vitro to model in vivo responses and future interventions; and 3) Test long-term safety and efficacy of engineered scaffolds and allografts with 4-AP +/- ES in a critical-sized sciatic nerve defect. Engineered repair of large-gap PNI using bioactive electrical and chemical cues will broadly impact the field. These studies will bridge the knowledge gap between the complex ES- mediated cell-material interaction microenvironment and poorly studied underlying regeneration pathways. These findings may improve the treatment of nerve defects, and inform exploratory work on regenerative strategies for innervation in other musculoskeletal tissues.

NIH Research Projects · FY 2026 · 2023-12

Peripheral arterial disease (PAD) affects 8 to 12 million people in the United States. Intermittent claudication (exercise-induced leg pain and severe walking limitation) is a hallmark of PAD which severely limits exercise capacity and deceases quality of life. Clinical studies demonstrate that the exercise pressor reflex (EPR), a neural reflex originating in skeletal muscle which contributes to the regulation of cardiovascular function during physical activity, is exaggerated in patients with PAD. Furthermore, the exaggerated EPR during exercise in PAD patients is seen before the onset of claudication. We hypothesize that in PAD, exercise-induced activation of muscle afferents (i.e. the EPR), which is initially below the threshold for pain , initiates an exaggerated vasoconstriction and worsens the ischemia of the exercising PAD muscle, which may further activate muscle afferents above the nociceptive threshold level and produce the familiar exertional pain symptoms of claudication. In Aim 1, We will examine the time-course of EPR function including heart rate, blood pressure and local muscle blood flow during static and dynamic exercise in a rat model of chronic PAD and in sham animals. We will determine the potential correlation between EPR function, muscle ischemia and pain behavior scores in both sham and PAD rats. Finally, we will determine if selective ablation of the EPR with the potent TRPV1 receptor agonist, resiniferatoxin (RTX), improves muscle perfusion during exercise and pain indices in PAD rats. Assessment of RTX efficacy will be carried out using a novel tissue clearing method in a TRPV1-tdTomato reporter mouse model. Furthermore, our pilot study found that neural inflammation indicated by macrophage infiltration occurs in lumbar DRGs post PAD. In parallel, we observed upregulated protein expression of nuclear factor erythroid 2-related factor (Nrf2, a transcription factor that mediates the cellular response to oxidative stress/inflammation) in lumbar DRGs post PAD. We believe that the purpose of upregulated Nrf2 protein is to counteract the influence of neural inflammation in DRG neurons post PAD. However, this self-limited compensatory response by an intrinsic DRG system may not be potent enough in DRG neurons to antagonize exogenous neural inflammation evoked by PAD. Therefore, in Aim 2, We hypothesize that selective upregulation of muscle afferent Nrf2 via Keap1-Knock-out (KO) will mitigate enhanced muscle afferent neuronal excitability and pain sensation in a mouse model of chronic PAD. We also hypothesize that local epidural delivery of hydrogel-based drug system with encapsulation of Nrf2 activator (i.e., Curcumin) will mitigate the exaggerated EPR and pain sensation in a rat model of chronic PAD. Finally, we propose to examine the potential influence of Nrf2 signaling onto DRG ion channel function such as potassium channels in muscle afferent neurons in PAD. To date, the underlying molecular/cellular mechanisms of the exaggerated EPR in chronic PAD has not been examined in any chronic PAD animal model. The long-term goal of this application is to discover the neural mechanisms operating in the exaggerated EPR and claudication and to provide a novel treatment for PAD.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Marijuana use among youth has been increasing, with considerably enlarged disparities across racial/ethnic groups. Racial minorities like African American (AA) adolescents are more likely to report marijuana use than their White peers. Marijuana use during adolescence can harm brain development and lead to other substance abuse and adverse health outcomes. In order to identify potential targets for interventions to reduce health disparities, there is a critical need to distinguish risk and protective factors for marijuana use that are specific to race and ethnicity. With a shifting legal environment for marijuana use in the United States and more adults supporting marijuana legalization, it is estimated that 8.2% of mothers and 9.6% of fathers living with adolescent offspring reported past-year marijuana use. The family environment is a major driver of youth development, and racial and ethnic families often experience greater exposure to systemic oppression (e.g., institutional and interpersonal racism, bias, and discrimination) and higher social inequality than their White peers, resulting in acute and chronic stress. Therefore, we hypothesize that parental marijuana use independently predicts offspring marijuana and other substance use, and intergenerational effects of marijuana use may be stronger among racial and ethnic families, especially among AA families. The overarching goal of this mixed-methods sequential explanatory study is to assess racial disparities in relationships between parental marijuana use and youth substance use. In Aim 1, we will leverage a longitudinally national survey with 5 waves of parent and offspring data (n≈3,272) to examine the intergenerational association between G1 marijuana use and subsequent G2 marijuana, vaping, tobacco, and other illicit drug use across distinct racial/ethnic groups (AAs, H/Ls vs. non-Hispanic Whites). In Aim 2, we will assess moderation and mediation effects from marijuana legalization status and other determinants (e.g., discrimination and psychological distress) across racial/ethnic groups. In Aim 3, we will conduct semi- structured interviews of parent marijuana users and their offspring (n≈96 each) to qualitatively examine the intersections of parent influences and policy change on youth substance use. Analyses of intergenerational and longitudinal surveys and semi-structured interviews will be fully integrated to complement each other and provide more robust findings. This study is innovative in its focus on assessing racial disparities in the intergenerational transmission of marijuana use in AAs and H/Ls, two NIH-designated health disparity populations. This study is also timely in responding to the rapidly shifting policy environment, rising marijuana use among youth and adults, and growing disparities by racial/ethnic group, with unknown etiology on the intergenerational drug use by racial/ethnic groups. The findings will inform the development of a future R01 to test tailored strategies to prevent youth marijuana and other substance use and reduce health disparities. 1

NIH Research Projects · FY 2025 · 2023-09

According to numerous in vitro studies, formation of amyloid beta (Ab) and other amyloidogenic protein is a self-assembly process that results in the production of neurotoxic oligomeric aggregates along with fibrillar structures, a hallmark of diseases such as Alzheimer’s disease. However, the vast majority of in vitro studies are performed at Ab concentrations of several orders higher than the physiologically relevant concentrations of Aβ in the brain; no aggregation of Aβ is observed at the low nanomolar concentration found in vivo. This suggests that the assembly of Aβ in aggregates in vivo utilizes pathways different from those used in experiments in vitro. We discovered that spontaneous assembly of Aβ42 oligomers from monomers within the physiologically relevant concentration range can occur by utilizing the on-surface aggregation mechanism. Here, the surface acts as a catalyst for the aggregation process. We developed a model that explains the surface catalytic effect of the amyloid aggregation from monomers at low nanomolar concentrations. Our central hypothesis is that the self-assembly of Aβ oligomers is initiated by the interaction of amyloid proteins with the cellular membrane. The membrane effectively catalyzes amyloid aggregation by stabilizing aggregation-prone conformations of amyloids. A thorough testing of this hypothesis is the major goal of this application. The rationale for the proposed goal is that understanding the fundamental mechanisms of membrane-mediated aggregation will guide the development of practical approaches to control the aggregation process. The objective of this proposal is to characterize the on-surface formation of Aβ oligomers, identify the aggregation-prone composition of cellular membranes, and develop a molecular model for future use in translational studies. Guided by strong preliminary data, we will test our central hypothesis through the following three specific aims: Aim 1: Characterize the aggregation process of Ab monomers catalyzed by cellular membranes with different lipid compositions. Aim 2: Evaluate contributions of free lipids of cell membranes into the membrane catalysis of amyloid aggregation. Aim 3: Develop a molecular model for the membrane catalysis phenomenon using theoretical and computational approaches. Aim 1 is focused on testing our hypothesis that the lipid composition of the membrane bilayer is the defining factor in spontaneous aggregation of Ab proteins at physiological concentrations. Under Aim 2, we will test the hypothesis that free lipids contribute further to the membrane catalysis of amyloid aggregation. Aim 3 proposes the use of theoretical approaches and high-power computer modeling to gain structural insights into the molecular mechanism behind catalysis of aggregation by the cellular membrane. The predictions of the theory will be tested under Aims 1 and 2, and the experimental results obtained from these Aims will be further used to tune the theoretical methods. The proposed research plan, combining experimental studies with extensive computational modeling, will provide a molecular model for the aggregation process catalyzed by membranes at physiological concentrations of monomers. The development of potential preventions for the interaction of monomeric amyloids with membrane can help to control the aggregation process. This is a paradigm shift, which opens prospects for the development of efficient treatments, early diagnostics, and preventive therapies for Alzheimer's disease.

NIH Research Projects · FY 2025 · 2023-09

Abstract: Cell motility is required for in situ cancer to form invasive primary prostate cancer (PCa). Inhibiting motility would intercept formation of invasive cancer. Until now it has not been possible to selectively inhibit motility. We have discovered a precision acting first-in-class agent, KBU2046. It selectively inhibits motility, has a wide therapeutic window, has led us to uncover heretofore unknown regulatory pathways, targets those pathways, performs well in GLP grade IND enabling studies, and opens up new high value innovative concepts for cancer prevention-interception. Studies support the hypothesis that KBU2046 is an effective cancer interception agent that operates through a novel mechanism, and that it and the pathways it modulates will have high potential for inhibiting the development of invasive cancer, which in turn has lethal potential. Aim 1. Identify and validate KBU2046’s pharmacologic binding site. In silico, biophysical and biochemical studies suggest that KBU2046 binds at the protein-protein interface between HSP90β and CDC37 and selectively decreases binding to Raf1. Using KBU2046-based photo-crosslinkable probes and proteolysis targeting chimeras (PROTACs), coupled to chemo-proteomic approaches and bioinformatic analysis, studies will identify and validate KBU2046 binding sites in the cellular milieu. Aim 2. Characterize the signaling pathway by which Raf1 regulates the motility of human prostate cells. We show KBU2046 decreases phosphorylation of ser338 on Raf1’s activation motif, that downstream regulation of motility is mediated through a novel pathway wherein Raf1 forms a complex with the actin binding protein, L- plastin (LCP1), while mass spectrometer (MS) proteomic / bioinformatic analysis demonstrates primary effects on cell motility proteins. Studies will examine this pathway in at risk human prostate cells, will use Raf1 active/inactive constructs, similarly engineer LCP1 constructs, probe signaling through orthogonal analytics, profile effects on protein signature using MS, and will examine the role of individual pathway members on cell motility and KBU2046 efficacy. Aim 3. Evaluate targeting motiliy in clinically relevant models of human disease. In the RapidCaP transgenic model, codeletion of Pten and p53 induces in situ→invasive→metastatic PCa. It supports treatment before versus after development of in situ lesions, allowing us to examine prevention and interception efficacy, respectively, for inhibiting formation of invasive cancer. Using primary cells from our prostate rapid autopsy program (RAP), we show mechanistically relevant responsiveness to KBU2046. Primary cells and resultant organoids provide a unique opportunity to examine efficacy in clinically relevant models. RapidCaP and RAP tissues will be interrogated at molecular and functional levels to examine effects of KBU2046. Impact. Investigators seek to advance the innovative concept that cell motility is an important cancer prevention-interception target. It has applicability to many cancer types.

NIH Research Projects · FY 2023 · 2023-09

Abstract Dementia is a major challenge to public health without effective therapy. Ambient fine particulate matter (PM) exposure significantly increases the risk for cognitive dysfunction and dementia with the mechanism(s) largely unknown. Animal studies have revealed that PM exposure significantly impairs cognition with decreased learning capability without clearly defined mechanisms. Small cerebrovascular diseases significantly contribute to vascular cognitive impairment and dementia. Preliminary studies showed that PM exposure resulted in significant small cerebrovascular inflammation with excessive reactive oxygen species (ROS) production in association with significant increases in serum TNF-α, IL-6, and IL-1β in male wildtype (WT) C57BL/6 mice. While PM exposure also induced significant cerebral vascular inflammation in female WT C57BL/6 mice, only IL-6 and IL-1β, not TNF-α, were significantly increased. Treatment of male WT mice with TNF-α, but not IL-6 or IL-1β, induced significant inflammation in small cerebral vasculature, and no inflammation was observed in small cerebral vessels in male TNF-α deficient mice with PM exposure. In contrast, treatment of female WT mice with IL-6 triggered a significant inflammation in cerebral vasculature, and no inflammation was present in cerebral vessels in female IL-6 deficient mice with PM exposure. Initial data also demonstrated that PM exposure significantly impaired the memory and learning capability in male mice that were effectively prevented with TNF-α deficiency (either with specific antibody treatment or TNF-α knockout). Thus, the present project was proposed to test the novel hypothesis that “PM exposure induces significant small cerebrovascular inflammation due to increased production of TNF-α in males and IL-6 in females, leading to cognitive dysfunction”. There are three specific aims: 1) to test the hypothesis that TNF-α mediates the effect of PM exposure on small cerebrovascular inflammation in males; 2) to test the hypothesis that IL-6 plays a critical role in PM exposure-induced small cerebrovascular inflammation in females; and 3) to investigate the mechanisms for sex differences in PM exposure-induced productions of TNF-α and IL-6. Aging WT C57BL/6 mice (both male and female) will be exposed to PM. ROS production and cerebral microvascular inflammation will be quantitatively evaluated at different time points after PM exposure. Brain MRI imaging will be performed to evaluate structural changes at baseline, 1 week (acute effect), and 2 months (chronic effect) after PM exposure. Mouse learning and memory capability will be longitudinally evaluated at baseline, 2, 4, and 8 weeks after PM exposure. Studies will also be conducted using male TNF-α deficient mice and female IL-6 receptor deficient mice, as well as macrophage depletion mouse model (both male and female) to define the mechanism(s). The data from the present project will provide important and novel information on the mechanisms for the development and progression of dementia in patients with PM exposure and help explore new approaches to preventing and treating dementia related to PM exposure.

NIH Research Projects · FY 2024 · 2023-09

Abstract The link between prostate cancer (PCa) risk and alcohol consumption has long been debated. We recently found in an extensive assessment of the epidemiologic data that, since the onset of the PSA testing era, most studies indicate alcohol consumption is strongly associated with PCa risk. Despite this epidemiologic support, little is known about the underlying mechanisms linking alcohol to PCa risk, progression, and mortality. Dr. Petrosyan introduced the concept of "onco-Golgi," where the Golgi becomes fragmented, resulting in altered glycosylation and trafficking of glycoproteins. Additionally, this Golgi disorganization is linked to activating transcription factor 6 (ATF6)-mediated Endoplasmic Reticulum (ER) stress. This results in increased plasma membrane (PM) expression of αv integrins abnormally glycosylated by Golgi glycosyltransferase, N- acetylglucosaminyltransferase-V (MGAT5). Importantly, these MGAT5-modified integrins form clusters with pentameric Galectin-3, resulting in increased retention on the PM. This, in turn, modulates tumor cell behavior, including adhesion and migration, which promotes prostate tumor dissemination to lymph nodes and distant organs. We have found that ethanol (EtOH) treatment aggravates Golgi disorganization and PCa tissues from heavy alcoholics have higher MGAT5 expression and PM Integrin αv levels. We propose that EtOH promotes PCa lethality by increasing Integrin αv-mediated PCa progression. Altered glycosylation of Integrin αv in the onco- Golgi is expected to be exacerbated by alcohol's disorganizing effect on Golgi. Preliminary data demonstrate a positive correlation between the number of Golgi fragments and the PM intensity of Integrin αv in EtOH-treated docetaxel-resistant PCa cells. Importantly, we have found that PCa Golgi disorganization is autophagy-driven and that the autophagy inhibitor, Hydroxychloroquine (HCQ), restores the compact Golgi in advanced PCa cells. We have also shown that ATF6-mediated ER stress results in expression of underglycosylated, high-Mannose (high-Man) integrins to the PM through ER-PM junctions. Depletion of ATF6 decreases the amount of high-Man integrins on the PM and in ER-PM junctions, as well as reduces the total number of ER-PM communications. We have demonstrated the synergistic effect of Golgi restoration by HCQ and ER stress inhibition by ATF6 depletion in preventing orthotopic tumor growth and metastasis. These exciting data led us to expect that similar combination treatments will decrease the pro-metastatic effects of EtOH administration. We have observed rescued Golgi morphology in cells treated with both EtOH and HCQ, suggesting that Golgiphagy, blocked by HCQ, is involved in EtOH-induced Golgi disorganization. Additionally, HCQ restores the level of PM Integrin αv to that of control cells. We expect that combined HCQ treatment and ATF6 depletion will effectively attenuate the effects of EtOH on PCa aggressiveness and metastasis. Overall, these data will shed light on the previously unknown mechanism of alcohol-promoted prostate tumor growth and metastasis and provide a potentially effective therapeutic strategy.