University Of Nebraska Medical Center

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.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Ventricular arrhythmia is the leading cause of death for chronic heart failure (CHF) patients. Although the therapeutic potential of renal denervation (RDN) for ventricular arrhythmias has been reported extensively, RDN- induced adverse complications severely limit its use in the clinic. Our recent study revealed that macrophage expansion and neuroinflammation in the stellate ganglion (SG) contribute to CHF-increased cell excitability of cardiac sympathetic postganglionic (CSP) neurons, which subsequently promotes cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF rats. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a crucial mediator in macrophage activation. Our pilot data showed that RDN attenuates cardiac sympathetic overactivation and ventricular arrhythmias, which are accompanied by the marked reduction of GM- CSF level and macrophage activation in SGs in CHF rats. However, it remains unclear if the antiarrhythmic effect of RDN is achieved via attenuating GM-CSF-mediated inflammatory pathways in SGs in CHF. Following the discovery of the antiarrhythmic mechanisms of RDN, this proposal aims to develop a novel clinical intervention to achieve the therapeutic role of RDN and avoid its limitations. Since sympathetic innervation of the kidney originates primarily from neurons in the aorticorenal ganglion (ARG), targeting ARG neurons could be a logical therapeutic strategy for achieving the antiarrhythmic role of RDN. Considering the advantages of optogenetics, including rapid, specific control of neuronal activities by light-sensitive opsins, adeno-associated-virus- Archaerhodopsin (ArchT, an inhibitory light-sensitive opsin) gene will be transfected into ARG neurons in CHF rats. Specificity of neuronal expression of ArchT in ARG neurons will be achieved by linking a neuron-specific promoter to the ArchT gene. Continual optogenetic silencing in ARG neurons will be achieved by illuminating a LED probe that is controlled and powered wirelessly in freely moving animals. We hypothesize that optogenetic inhibition of ARG neurons would reduce CHF-elevated GM-CSF level in SGs, which subsequently alleviates macrophage activation and neuroinflammation in SGs, thereby attenuating CSP neuronal excitability, cardiac sympathetic overactivation, and ventricular arrhythmogenesis in CHF. Using multi-faceted technical approaches ranging from whole-animals to cellular-molecular levels, we will design in vivo and in vitro studies to assess these questions. Specific Aim 1, we will test if GM-CSF signaling axis contributes to macrophage activation and neuroinflammation in SGs from CHF animals. Specific Aim 2, we will address if GM-CSF signaling pathway contributes to CHF-increased cell excitability of CSP neurons, cardiac sympathetic overactivation, and ventricular arrhythmogenesis. Specific Aim 3, we will determine if optogenetic silencing in ARGs can achieve the antiarrhythmic effect of RDN by attenuating GM-CSF-induced macrophage activation and neuroinflammation in SGs in CHF. These studies will open a new avenue in therapeutics against lethal ventricular arrhythmia and provide a novel clinical intervention to reduce mortality and improve outcomes and quality of life in CHF patients.

NIH Research Projects · FY 2024 · 2023-08

Project Summary: Characterizing the Redoxome of Chlamydia and Its Host Cell 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 types. 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 pathogen 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 study chlamydiae mechanistically have significantly enhanced our understanding of this pathogen. This proposal applies a combination of these new genetic techniques and established mass spectrometry-based approaches to evaluate proteins subjected to redox regulation in both the host and bacterium. The hypothesis of the proposed work is that Chlamydia uses redox signaling to trigger secondary differentiation from the replicative to the infectious form of the organism. The major goals of this two-year funding proposal are to identify host and bacterial proteins subjected to redox regulation as a first step towards addressing our hypothesis. Selected wild-type and mutant proteins will be evaluated for changes in their redox status both in vitro and in cell culture. Results from this study will advance our understanding of this important pathogen and lead to further work to characterize the function of identified redox-regulated proteins. This may, in turn, lead to the design of novel therapeutic agents that are specific for Chlamydia. This will allow for minimal effects on normal flora for patients receiving treatment for this highly prevalent disease.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Chronic heart failure (CHF) has become epidemic in developed nations accounting for about 6.5 million patients in the US alone. Although the use of β-adrenergic blocking agents, ACE inhibitors and Angiotensin II receptor blockers have been highly effective in slowing the progression of the disease and reducing mortality, there remains an extremely high mortality and morbidity rate for patients diagnosed with CHF. In about half of patients with CHF, complex ventricular arrhythmias, including non-sustained ventricular tachycardia, are present and sudden cardiac death (SCD) is common. Abnormalities and alteration in cardiac sympathetic control of the heart are linked to life threatening arrhythmias, CHF and SCD. Current methods for chemical treatment of sympathetically mediated arrhythmias offer only short-term (i.e., lasting a few hours to one day) effect by temporarily blocking stellate ganglion (SG) neuronal activity with local anesthetics (i.e., SG blockade). Recurrent drug-resistant cardiac arrhythmia patients may be offered surgical stellate ganglionectomy to permanently remove part of the SG. Although this surgery is effective for removing cardiac arrhythmias, it is not the first choice of treatment recommended by cardiologists because of the invasive nature of this procedure. Here, we propose an innovative strategy to chemically ablate the SG function by blocking its surrounding vascular supply, thereby inducing sympathetic neuronal apoptosis and cell death. In our preliminary study, we developed an injectable hydrogel delivery system based on FDA approved biopolymers with encapsulating sunitinib (SU) loaded microspheres. SU is an FDA approved small molecule that has anti-VEGF receptor and other tyrosine kinase activities for patients with neuroendocrine tumors. We demonstrated that SU could be sustained released from our delivery system and the released SU could disrupt the in vitro angiogenesis and in vivo vascular bed after injection into the rat SG. We thus hypothesize that sustained released SU disables the SG function by disrupting its vascular supply and subsequently reverses the CHF-associated cardiac arrhythmia (Aim 1) and regulates cardiac remodeling (Aim 2). This application will use highly integrative techniques to evaluate the therapeutic efficacy of SU loaded delivery system, including novel Rosa-tdTomato flox/flox::Tie2 Cre reporter mouse model, tissue clearance technique, molecular biological techniques and whole animal experiments (in vivo conscious electrocardiogram telemetry recording, cardiac electrical mapping, pressure-volume loop analysis). We believe that this proposed research will lay a solid scientific and technological foundation for developing a new therapy for the patients with CHF and other cardiomyopathy and improve the quality of life of these patients.

NIH Research Projects · FY 2025 · 2023-08

Chronic heart failure (CHF) is one of the leading causes of death in the U.S. A primary characteristic of this disease is elevated sympatho-excitation and exercise intolerance during physical activity. During exercise in heart failure patients, extreme activation of the sympathetic nervous system is often seen and evokes an exaggerated pressor response accompanied by hyperventilation. These abnormalities potentially increase cardiovascular risk during physical activity in these patients. Experimental evidence suggests that 1) the exaggerated sympatho-excitation during exercise is directly related to an increased sensitivity of the exercise pressor reflex (EPR); 2) the enhanced mechanically sensitive afferent component of this reflex (i.e. mechanoreflex) primarily contributes to the exaggerated EPR in CHF and 3) muscle metaboreflex activated by femoral intra-arterial injection of capsaicin is blunted in CHF rats, which is associated with downregulated Transient Receptor Potential Vanilloid Type 1 (TRPV1) protein expression in lumbar dorsal root ganglia (DRGs). The molecular and cellular mechanisms underlying altered mechano- and metabo-sensitive afferent limb in CHF have not been fully understood. Our preliminary data showed that myocardial infarct (MI) triggered time-dependent macrophage infiltration into lumbar DRGs, suggesting that a neural inflammatory cascade occurs in muscle afferent ganglia post MI. We hypothesize that macrophage activation in lumbar DRGs plays a critical role in muscle afferent sensitization as well as the exaggerated EPR via regulating Kv channels and glutamatergic signaling in CHF. We also hypothesize that macrophage activation in lumbar DRGs also serves as an upstream mechanism to cause the TRPV1 channel dysfunction in muscle metabo-sensitive neurons in CHF. In Aim 1, we propose to determine the time-dependent macrophage infiltration/activation in lumbar (L4-L6) DRGs in post-MI male and female rats as well as post-MI CX3CR1CreER-tdTomato reporter mice. We will also plan to identify the pro-inflammatory (M1)/anti-inflammatory (M2) phenotypes of macrophages in lumbar DRGs post MI. Finally we will determine if pharmacological macrophage inhibition in lumbar DRGs can restore the exaggerated EPR as well as muscle afferent sensitization in CHF male and female rats. Aim 2 is designed to address how macrophages influence muscle afferent neuronal excitability post MI. We will determine if local pharmacological macrophage inhibition in lumbar DRGs can restore altered Kv channels, TRPV1 channel and glutamatergic dysfunction in CHF male and female rats. We will use highly integrative techniques including molecular (real-time PCR, western blot, immunofluorescence and tissue clearance), cellular (patch clamp) and whole animal experiments (measuring EPR function, single afferent recording) to test our hypotheses in this project. We believe that this proposed research will address important functional and mechanistic issues that directly relate to the quality of life in patients with CHF. These data will uncover new targets for therapy in this patient population.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT Phosphoinositides (PIs) are a minor class of phospholipids often comprising less than 1% of the cellular lipid cohort. Despite the low abundance, PIs have huge impacts on cell physiology and alterations of PI signaling pathways are associated with the pathogenesis of many human diseases including neurodegenerative diseases, metabolic disorders, autoimmunity, and cancer. This pathophysiological importance is largely due to the signaling roles of PIs which depend on the subcellular distribution of PIs and on key interactions between PIs and PI effectors. A twist in PI signaling is that in contrast to general belief, a substantial fraction of PIs is found in non-membranous nuclear compartments. The nature and functions of nuclear PIs remains largely unknown due to the lack of systematic studies of nuclear PIs and PI effectors. We have expertise in defining and characterizing novel PI effector proteins involved in key signaling pathways including vesicular trafficking, cytoskeleton dynamics, and transcription regulation. Since PI kinases are often associated with PI effectors thus ensuring PI generation is spatiotemporally linked to PI effector activation, we have performed proteomic analyses to identify the interactomes of the nuclear PI-generating kinases. Out of these proteomic screens, we have validated several nuclear complexes that associate with nuclear PI kinases or with PIs themselves. The validated complexes include transcription factors and coactivators, epigenetic enzymes and associated corepressors, the DNA repair machinery, and factors involved in RNA processing. We recently discovered that nuclear PIs accumulate at distinct subnuclear regions such as nuclear speckles and DNA double-strand breaks. Based on our novel discoveries of PIs and PI kinases interacting with effectors in the nucleus, the overarching goal of my research program is to decipher the signaling pathways emanating from the nuclear PIs. Our overall hypothesis is that upon suitable stimuli the activation of nuclear PI kinase at specific subnuclear compartments elevates the local concentration of nuclear PIs and these nuclear PI foci function as platforms to regulate PI effectors recruited to the foci mediating transcription regulation and assembly of complexes that regulate epigenetic changes. The goals of my research programs for the next five years include dissecting the nature and subnuclear distribution of nuclear PIs using novel microscopic tools which will enable us to obtain high resolution images of the nuclear PIs, defining the new roles of PIs regulating chromatin positioning to nuclear speckles, and investigating novel roles of nuclear PIs in regulating gene expression with focuses on transcription regulation and epigenetic repression with innovative cell biological, genome-wide, and biochemical approaches. Upon the completion of the research programs, we will obtain insight into the unexpected roles and molecular mechanism of PIs in the nucleus, with the goal of identifying novel strategies for targeting the nuclear PI signaling pathways dysregulated in diverse human disease.

NIH Research Projects · FY 2026 · 2023-07

Project Summary/Abstract Lipids provide a rich source of energy to fuel fundamental cellular processes including migration, differentiation, and survival through periods of low nutrient availability. The storage and utilization of lipid fuel relies on lipid droplets (LD). These unique organelles are surrounded by peripheral phospholipid monolayer that encapsulate a core containing neutral lipids triacylglycerol (TAG) and cholesterol ester (CE). LDs possess a unique proteome of adapters, enzymes, and structural proteins that interact directly with the LD monolayer and regulate trafficking for biogenesis and catabolism. However, detailed mechanistic knowledge regarding the relationship between the LD monolayer and LD trafficking machinery is lacking. This knowledge is especially critical for the process of lipophagy, whereby subpopulations of LDs are selectively targeted for lysosomal degradation. Our preliminary data suggest that LDs possess heterogenous lipid signatures at the level of the LD monolayer, and this heterogeneity influences the fate of certain small LD subpopulations toward lipophagy. Furthermore, we postulate that lipids of the LD monolayer including diacylglycerol (DAG) and phosphatidtyliositol (PI) are modulated by LD catabolic enzymes to influence their trafficking. In Project 1, we will determine the role of adipose triglyceride lipase (ATGL) in generating DAG on the LD surface via TAG hydrolysis, and explore the impact of DAG on the recruitment and activation of protein kinase C (PKC) on the LD surface. In Project 2, we will investigate Rab5 in recruiting PI 3-Kinases to the LD surface to phosphorylate PI, define the role of ESCRT proteins in the trafficking of LDs for lipophagy in mammalian cells. We will also explore an unexpected role for certain ESCRTs in LD biogenesis. The results gained from the proposed research will provide a mechanistic understanding of lipid droplet trafficking and the modulation of the LD monolayer.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT People who experience repeated brain injuries (BIs) before the brain recovers from the initial trauma are at a high risk for neurological deficits, depression, suicide, and Alzheimer-like symptoms. Women who experience intimate partner violence (IPV) are at high risk for injuries to their head, neck and face – potentially resulting in an acquired BI. Among IPV survivors, BI can occur due to a traumatic brain injury (TBI) resulting from a bump, blow, or jolt to the head or through strangulation. BI can have long term consequences on IPV survivors' well- being and quality of life. Comprehensive screening among this vulnerable population and timely neurorehabilitation interventions (i.e., referral to, and receipt of, evidence-based approaches to mitigate BI- related health outcomes) have the potential to mitigate the impact of IPV-related BI and improve health outcomes. Despite the high risk of BI among IPV survivors and the availability of evidence-based approaches to address BI, there are no standard guidelines for BI screening in this group of women. The long-term goal is to advance scientific understanding of implementation strategies related to the secondary prevention of IPV- related BI that will inform future policies for timely screening, referral, and neurorehabilitation and ultimately reduce neurological damage among this population. The objective of this project is to examine how screening and detection of IPV-related BI can be effectively integrated into the workflow for community-based organizations (CBOs) serving IPV survivors using a participatory approach. The central hypothesis has been formulated based on preliminary studies and posits that CBO and stakeholder derived implementation strategies can improve the adoption, implementation, and maintenance of timely screening/detection and referral of IPV survivors at risk for a BI. Using the PRISM/RE-AIM framework and a participatory approach of community-engaged dissemination and implementation, these aims will be pursued. Aim 1: To evaluate adoption, implementation, and sustainability strategies of a brain injury screening intervention of IPV survivors at CBOs with and without stakeholder developed implementation strategies. Aim 2. To determine the reach and effectiveness of a brain injury screening intervention at CBOs serving survivors of IPV. Exploratory Aim. Determine client and CBO implementer's perception of the screening intervention and implementation strategies used in the project. The expected outcome is to increase currently limited knowledge of potential implementation strategies to integrate routine screening and referral for IPV-related BI. This outcome is expected to significantly contribute to the knowledge base needed to fully integrate the routine screening and referral of IPV-related BI in CBOs serving IPV survivors. The project will also allow for the identification of potential screening and referral strategies that can be used to connect patients with IPV-related BI to evidence- based approaches to mitigate poor health outcomes and identify methods to achieve secondary prevention.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY In the US, over one-third of older adults ≥60 years with acute myeloid leukemia (AML) die without receiving any chemotherapy, primarily due to concerns of cognitive decline and loss of functional independence if they chose to undergo intensive chemotherapy. A combination of venetoclax and low-intensity chemotherapy offers an effective new treatment for AML. However, several knowledge gaps exist about this new approach. Venetoclax targets neuroprotective B-cell lymphoma 2 (Bcl-2) proteins, but the actual risk of cognitive decline and loss of functional independence following its use, and the factors that increase such risks, are still unclear. Our preliminary data demonstrated that many older adults with AML had stable or improved cognitive and physical function, functional independence, and health-related quality of life (HRQOL) from the time of diagnosis to three months following newer treatments. These novel findings highlight the success of newer treatments in improving cognitive function, functional independence, and HRQOL. Despite this, frailty and multimorbidity were identified as risk factors for functional decline. We also utilized the NCI-recommended neuroscience approach and conducted electroencephalography (EEG)/event-related brain potential (ERP) studies. In our study, adults with hematological cancers versus healthy adults demonstrated altered activity in the dorsal attention and central executive brain networks prior to treatment. Confirmation of these findings will identify the risk of a decline in attention and executive function, functional independence, and HRQOL, and will provide novel mechanistic insights into the activity of brain networks in older adults with AML. In the proposed longitudinal cohort study, we will enroll older adults with a new diagnosis of AML who will receive venetoclax-based treatments, and age-, sex-, and education-matched non-cancer controls. We will compare the two groups on the following outcomes: attention and executive function (Trail Making and other neuropsychological tests), functional independence, HRQOL, and EEG/ERP measures (ERP studies during auditory-visual distraction tasks to measure brain activity) at enrollment and over 12 months. The study aims are: Aim 1. Determine the longitudinal attention and executive function of older adults with AML before and after venetoclax-based treatment, as compared to age-, sex-, and education-matched non-cancer controls. Aim 2. Determine longitudinal changes in functional independence and HRQOL in older adults with AML versus controls, and examine their associations with longitudinal changes in attention and executive function. Aim 3. Measure longitudinal changes in the activity of brain networks of older adults with AML before and after venetoclax-based treatment, as compared to controls. This is the first study to apply rigorous methods to overcome key limitations of prior studies and to advance the current limited cognitive aging research in patients with AML. Understanding factors associated with stable/improved functional trajectory following new treatment can change treatment paradigms and is essential to inform critical decision-making of older adults with AML considering chemotherapy for this fatal disease. The goals align with the NCI/NIA priorities.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY. Chronic, non-healing wounds are currently affecting more than 6 million Americans. They have significant impact on patients’ mobility and quality of life, and can lead to a high incidence of amputation and mortality rate. Biofilm infection is a critical factor that leads to chronic wound formation. Biofilm bacteria are very difficult to kill compared to planktonic bacteria due to their reduced growth and metabolic rates, the presence of persister cells, inducible resistance mechanisms in response to antibiotic challenges, and the mutational resistance development. Current clinical standard of care for chronic wound biofilm infections uses repeated debridement with prolonged systemic or topical administration of antimicrobial agents. This treatment has limited efficacy and imposes a significant burden on both patients and healthcare providers. The development of more effective delivery technologies for antimicrobial agents and physical biofilm treatment methods is a very active research area. However, current technologies reported in the literature offer limited improvement in anti-biofilm efficacy, may cause potential damage to host tissues, or require a long-term application to be effective. There is a critical need for more efficacious and safer biofilm treatment technologies that does not require long-duration and frequent treatment applications to facilitate a timely closure of chronic wounds. Our long-term goal is to apply engineering innovations and technological advances to providing better healthcare to chronic wound patients. Our overall objective in this proposal is to develop a novel, electric current-based system to provide a complete treatment strategy for multispecies chronic wound biofilm infections from the initial reduction of bacterial bioburden to the long-term maintenance of wound sterility during the entire course of wound healing. Our system will perform two functions to achieve this goal: 1) electrical debridement of biofilm by high- intensity electric current application; and 2) rapid delivery of high-concentration antibiotics and antimicrobial nanoparticles by high-intensity iontophoresis. The electrical debridement and antibiotics will achieve a rapid initial reduction of biofilm bacterial count to below the clinical threshold for wound infection (105 CFU/g). The antimicrobial nanoparticles will then maintain a low bacterial bioburden, prevent biofilm reformation and new infections throughout the wound healing process. Our proposed system will be based on a novel hydrogel ionic circuit technology developed in our lab to allow safe application of high-intensity current to wound tissues to significantly enhance electrical debridement efficacy and iontophoretic delivery efficiency for antibiotics and antimicrobial nanoparticles. If successful, our biofilm treatment system will have direct positive impact on all patients suffering from chronic wounds by significantly reducing the wound healing duration, the amputation rate and mortality rate associated with chronic wounds.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Tumor Initiating Cells (TICs) are a subpopulation of tumor cells defined by their ability to self-renew and regenerate the heterogeneous tumor. TICs have altered metabolic and drug efflux properties, allowing them to persist as a sanctuary population of resistant cells during treatment. As such, TICs mediate tumor recurrence and metastasis, which are key contributors to mortality. To date, however, there are no therapies targeting the TIC population in solid tumors. My data show that the formation and maintenance of TICs in Ras-driven colorectal and lung tumor cell lines are critically dependent on the molecular scaffold Kinase Suppressor of Ras 1 (KSR1). KSR1 coordinates signaling through the Raf-MEK-ERK cascade downstream of oncogenic Ras. KSR1 is necessary for Ras-driven tumor formation, but dispensable for normal cell growth. Moreover, ksr1-/- mice are phenotypically normal but resistant to cancer formation. These characteristics highlight the value of KSR1 as a potential therapeutic target. Our data further show that KSR1 KO can prevent MEK inhibitor trametinib-mediated expansion of the TIC population and restore sensitivity to trametinib in both NSCLC and in CRC where resistance currently limits clinical utility of this drug. Further characterization using KSR1 transgenes with defined mutations in downstream effector-binding regions has identified KSR1 interaction with both ERK and AMPK to be implicated in maintenance of the TIC population, in vitro clonogenicity, and resistance to trametinib. These data suggest the hypothesis that KSR1-dependent ERK and AMPK activation in Ras-driven cancers plays a selective role in the formation of tumor-initiating cells and the drug resistance conveyed by this subpopulation. I will test this hypothesis by 1) defining the proximal KSR1-dependent signaling pathways that control TICs and trametinib resistance; and 2) assessing the ability of targeted KSR1 degradation to impair tumor-initiating capacity in a preclinical tumor organoid model. Completion of these aims will expand mechanistic understanding of Ras-driven TIC formation and maintenance and may reveal a novel therapeutic approach to treat TIC-mediated resistance, recurrence, and metastasis.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract Psychiatric disorders are the most prevalent debilitating illnesses across the lifespan. Importantly, epidemiologic studies indicate that 75% of all diagnosable psychiatric disorders begin prior to age 24, highlighting the need for investigations of the developing brain. Exposure to traumatic events during childhood and adolescence can induce maladaptive functioning of the hypothalamic-pituitary-adrenal (HPA) axis, leading to long-term structural and functional alterations in stress-sensitive brain regions that are known to be critical for cognitive control. Similar neural aberrations have been identified across a range of mental health disorders, including anxiety, ADHD, and depression. However, the extant literature focusing specifically on the impact of trauma and HPA axis activation on the development of cognitive control networks is extremely sparse. Given the high incidence of trauma in youth and ties with psychopathology, it is imperative that we develop a more detailed understanding of the neurophysiological changes underlying brain development in the context of trauma exposure. The proposed fellowship aims to partially remedy these knowledge gaps by utilizing the excellent spatial and temporal precision (i.e., millisecond) of magnetoencephalography (MEG) to investigate the developmental trajectory of brain networks critical to cognitive control and the impact of trauma and HPA axis activation on such networks. Briefly, participants between the ages of 6 to 15 years-old will annually complete a cognitive control task during MEG, complete cognitive and emotional assessments, and provide hair samples. The resulting MEG data will be transformed into the time-frequency domain and imaged using a beamforming approach, while the hair samples will be used to compute mean cortisol concentrations over 3 months. The output dynamic functional maps will be used to examine spectrally specific brain responses that are developmentally-sensitive in regions critical to cognitive control. Aim 1 will map the longitudinal development of neural dynamics and network connectivity that underlie response selection in typically-developing youth from age 6 to 15 years-old. Aim 2 will determine the impact of trauma exposure and HPA axis activation on the longitudinal development of cognitive control networks, neuropsychological performance, and emotional health. To this end, we will leverage the latest MEG and source reconstruction techniques, neural oscillatory dynamics and connectivity analysis methods, hair cortisol concentrations, and cognitive assessments to map variability in the neurophysiological bases of cognitive control development in healthy youth. In addition to the sponsor, who is a developmental cognitive neuroscientist with extensive mentoring and NIH funding experience, we will leverage an expert mentoring team comprised of leaders in cortisol analysis (Dr. Megan Gunnar), trauma assessment (Dr. Patrick Tyler), and the neuronal impact of childhood adversity (Dr. Christopher Monk). In summary, this research will aid in providing a more complete foundational understanding of the key neurological processes underlying cognitive control perturbations in youth with trauma and identify potential windows for intervention.

NIH Research Projects · FY 2025 · 2023-04

A significant portion of the global burden of AUD (Alcohol use disorder) derives from an increased risk and susceptibility to diseases including infection, cirrhosis, and cancer. These diseases are influenced by the composition of the intestinal microbiota, which is altered by alcohol use. Though it has been accepted that change to the microbiota contributes to disease establishment and progression in AUD, our understanding of the mechanisms by which this occurs is inadequate. These mechanisms likely involve a variety of microbiota- derived products (membrane vesicles, excreted chemicals, cellular components, LPS, etc.). The preliminary data addressed herein support the proposition that microbiota-derived membrane vesicles (MVs) are an important driver of alcohol-driven tissue injury. To address this possibility, this proposal will examine the effects of alcohol on the composition and frequency of microbiota derived MVs. This proposal will also evaluate the effects of alcohol-associated MVs on promoting tissue injury and mucosal infection. Our preliminary data demonstrate that alcohol alters the composition of MVs generated by the gut microbiota, and that isolated alcohol-associated MVs increase susceptibility to respiratory infection, independent of alcohol use, suggesting that MVs influence mucosal host defense. We hypothesize that MVs from an alcohol-associated microbiota will have increased inflammatory properties and increase susceptibility to mucosal infections via epithelial inflammation and barrier dysfunction. Understanding the alterations to bacterial MVs following alcohol exposure may give new insight into disease pathogenesis, which may inspire future therapies centered on modulating MVs.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Heart failure with preserved ejection fraction (HFpEF) is one of the greatest treatment challenges in cardiovascular care today. Exercise is one of few treatments shown to benefit adults with HFpEF. Yet, to achieve and sustain the benefits of exercise, adherence is required. Recent National Heart Lung and Blood Institute (NHLBI) working groups on HFpEF and exercise in heart failure highlighted high priority areas for future study including examining strategies and interventions to promote exercise initiation and adherence, testing interventional mechanisms to improve adherence to exercise, identifying clinically meaningful outcomes for heart failure trials beyond mortality, and examining longitudinal changes in inflammatory biomarkers to better understand correlates to clinical status. Our team has successfully tested an intervention [Heart Failure Exercise and Resistance Training (HEART) Camp] that significantly improves long-term adherence to moderate intensity exercise (≥120 minutes of exercise at a heart rate reserve of 40-80%) in stable, chronic heart failure. Adherence was moderated by ejection fraction and a secondary analysis of our HFpEF subgroup showed promising long- term exercise adherence. We now propose a sufficiently powered randomized controlled trial to test the efficacy of 2 interventions in achieving long-term exercise adherence in adults with HFpEF. Our overall objectives align with NHLBI priorities and work toward achieving our long-term goal to promote adherence to exercise in HFpEF: (a) evaluate the effects of theory-based training and coaching interventions on long-term adherence to exercise, (b) identify minutes of moderate-intensity exercise that relate to clinically meaningful change in patient-reported outcomes, (c) evaluate interventional mechanisms and interim clinical events as mediators of adherence behaviors, and (d) examine the cost of intervention delivery. To meet these objectives, we propose a 3-group randomized controlled trial to compare 2 interventions, HEART Camp (in-person) to HEART Camp Connect (virtual) to each other and to enhanced usual care in adults with HFpEF. The proposed study incorporates several innovations: 1. We are the first to: test the effects of behavioral interventions designed to promote long-term exercise adherence in HFpEF using an objective measure of adherence, attempt to define a benchmark of minutes of exercise needed to achieve a clinically meaningful change, and assess exercise intervention cost; and 2. We incorporate technical innovations including a web application for real-time capture of exercise data, an innovative analytic approach that examines the influence of interim clinical events on adherence, a HFpEF algorithm and large-scale inflammatory assays. These innovations challenge the current paradigm and help to reach new horizons in HFpEF science. Our approach, which combines well-studied theoretical mechanisms delivered with virtual coaching and training tested against in-person coaching and training, will allow us to better understand exercise adherence in HFpEF. This study will have a critical impact by lessening non-adherence as a barrier to progress in the clinical care of adults with HFpEF through regular exercise training.

NIH Research Projects · FY 2026 · 2023-04

PROJECT ABSTRACT Brain tissues from people living with HIV (PLWH) showed evidence of Alzheimer’s Disease (AD)-like pathologies, including increased neurotoxic amyloid-b (Ab40/42), amyloid plaques and Tau hyperphosphorylation (pTau) associated with neurodegeneration and HIV-associated neurocognitive disorders (HAND). The mechanisms through which HIV increase Ab, pTau, and induce AD-like central nervous system (CNS) impairment are not known. Although most PLWH with CNS Ab plaques and pTau had been on long-term antiretroviral therapy (ART), the role of ART in pTau and Ab production, and AD-like pathologies has not been investigated. Findings from our R21 studies, using in vitro, in vivo and ex vivo approaches, demonstrated that HIV-1 significantly increased CNS Ab42 and pTau in humanized mice, and this was associated with significantly 1) increased expression and activation/activity of b-secretase-1 (BACE1), soluble (s)APPb (amyloidogenic pathway effectors), and GSK3b (kinase that phosphorylates Tau); 2) reduced expression/activity of neprilysin (NEP, Ab-degrading enzyme); 3) increased blood-brain barrier (BBB) expression of the receptor for advanced glycation end products (RAGE, mediates Ab CNS influx), and reduced BBB expression of low-density lipoprotein receptor–related protein-1 (LRP1, mediates CNS Ab efflux); and 4) increased neuroinflammation, neuronal damage and BBB injury. We demonstrated that LRP1 and CCR5 mediated Ab transport through the BBB, that AZT potentiated HIV-induced upregulation/activation of BACE1, sAPPb and GSK3b, and that AZT blocked NEP expression/activity. Significantly, the CCR5 antagonist maraviroc (MVC) abrogated these HIV- and AZT- induced effects. Thus, we hypothesize that CCR5, via LRP1, plays a major role in Ab formation, transport, and catabolism, amyloidogenesis and pTau in PLWH, and that targeting CCR5 prevents HIV-induced Ab and pTau, increases Ab CNS efflux, and abrogates CNS injury and HAND. Using a validated HIV/AIDS animal model, primary human cells and human brain tissues, we will test this hypothesis and further investigate the effects of commonly prescribed antiretroviral (ARV) drugs (AZT, TDF) on HIV-induced activation of BACE1 and APP amyloidogenic pathway, Tau metabolism, and CNS injury (Aim-1); Ab transport, degradation, clearance, and neuroinflammation (Aim-2); and associated BBB transcriptomic and epigenomic signatures (Aim-3). These mechanistic studies will help determine whether HIV induces amyloidogenesis by i) activating BACE1 pathways to increase Ab production or ii) by interfering with LRP1-mediated Ab transport, degradation, clearance; iii) the role of ARVs; iv) whether CCR5 modulates these effects; and v) characterize the brain vascular transcriptome and epigenome associated with HIV/ARVs-induced dysregulation and MVC protective effects. Our proposed studies are of high-impact, translational significance, and address the NIH high priority research areas that focus on “Examining the pathophysiologic mechanisms of HIV-induced CNS dysfunction in the setting of ART…and development of novel therapeutic approaches to mitigate CNS complications of HIV infection.”

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Pancreatic cancer remains a lethal cancer in part because of our lack of understanding of its progression and pathogenesis. Dysregulation of normal progenitor cells can result in the initiation of cancer. An often-overlooked epithelial progenitor niche of the pancreas are the pancreatic ductal glands (PDGs), which are blind outpouches stemming from the main pancreatic duct. Previous analyses have shown an enrichment of pluripotent potential and pancreatic cancer pathways within cells of the PDGs. Cells expressing trefoil factor family 2 (TFF2) give rise to differentiated progeny that migrate to the ductal epithelium during pancreatitis; in-vivo TFF2 knockout experiments have shown these cells are crucial for regeneration of the ductal epithelium. The contribution of this progenitor niche to PDAC initiation and progression remains largely unexplored. Transgenic mice were developed in which expression of Cre-recombinase can be induced within TFF2 expressing cells (Tff2CreERT). Mice with Cre-dependent oncogenes and a dual-color fluorescent reporter (KrasLSL- G12D; P53fl/fl or Smad4fl/fl; Rosa26mT/mG) were crossed with these animals allowing for simultaneous activation of oncogenes and permanent GFP tagging of TFF2 expressing cells of the PDGs and subsequent progeny. Preliminary data show that TFF2-expressing PDG cells exhibit increased stemness following oncogenic activation and give rise to a wide spectrum of lineage traced premalignant lesions and cancers. Our central hypothesis is that the activity of oncogenic insults in PDG cells with stem-like properties results in unique lineage specification and transformation, producing premalignant and malignant lesions similar to those observed in humans.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Liver injury from alcohol abuse, and obesity can lead to liver inflammation, steatosis, and fibrosis. Liver cell damage results in the release of inflammatory cytokines from hepatocytes and Kupffer cells (KCs), which cause the activation of hepatic stellate cells (HSCs). If unresolved, it may result in liver fibrosis and progression to cirrhosis. Hedgehog (Hh) signaling regulates multiple pathways in liver fibrosis, including epithelial-to- mesenchymal transition (EMT), HSC activation, and inflammation. Further, chronic liver diseases are associated with the dysregulation of miRNAs. Specifically, miR-96 is upregulated after liver damage and promotes fatty liver disease (FLD). miR-96 caused malfunctioning of insulin receptor (INSR) and insulin receptor substrate (IRS)-1 results in impaired insulin signaling and glycogen synthesis. Further, miR-96 downregulates SMAD7 and FOXO1-3 and promotes transforming growth factor-beta 1 (TGF-β1) mediated liver fibrosis. In our preliminary studies, we demonstrated the upregulation of Hh signaling ligands including PTCH1, SHH, and GLI2 cause significant increase in collagen and fat deposition in 5% ethanol and high-fat diet (HFD) fed mouse. We synthesized a novel Hh pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis(2- pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) with GLI1/2 inhibitory activity in nanomolar (nM) concentration. Treatment of both HFD and alcohol-induced liver disease (ALD) mice with MDB5 resulted in a significant decrease in the levels of liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and further ablate GLI2, and its target genes. Our miRNA profiling in ALD and HFD mouse liver identified miR-96 was consistently upregulated. Target Scan analysis revealed that miR-96 targets several anti-inflammatory and anti-fibrogenic genes. The knockdown of miR-96 expression in hepatocytes and HSCs with anti-miR-96 resulted in the restoration of affected genes SMAD7 and FOXO3. We synthesized glycyrrhetinic acid (GA) conjugated GA-PEG-P(Asp)-g-DC-g-TEPA copolymers for liver-specific in vivo delivery of MDB5 and anti-miR-96, respectively. There was a significant increase in MDB5 concentration in the fibrotic liver at 1h post systemic administration of MDB5 loaded GA-NPs. TGF-β and Hh signaling crosstalk whereby TGF-β1 induces GLI-1 through downstream consequence of RAS signaling. Therefore, we hypothesize that the combination therapy of MDB5 and anti-miR-96 using GA-NPs could prevent alcohol and fat induced liver injury, and fibrosis. Our specific aims are to i) establish the therapeutic efficacy of the Hh inhibitor MDB5 on alcohol and high fat diet induced liver injury; ii) establish the profibrotic role of miR-96 in in HFD and ALD mouse models, and iii) determine the therapeutic efficacy of liver targeted NPs loaded with MDB5 and anti-miR-96 for treating HFD and ALD mouse models. Our long-term goal is to understand the progressive mechanisms of ALD and NAFLD to liver fibrosis, and establish new, liver-specific treatments.

NIH Research Projects · FY 2026 · 2023-02

The goal of the Alcohol Center of Research-Nebraska (ACORN) is to expand existing strengths and expertise in Nebraska to develop a novel niche in alcohol research, by creating a Center focused on the Alcohol Exposome. Alcohol research has been a major area of emphasis in Nebraska (at the Omaha VA Medical Center Research Service and at the University of Nebraska Medical Center) for over 5 decades. As a result, many contributions to the field of alcohol research and the treatment of alcoholic liver disease have been made. Studies examining the effects of alcohol on trace metal metabolism, lung biology and physiology, mechanisms of alcoholic fatty liver, role of aldehyde adducts in alcoholic cell injury, hepatic protein trafficking and signal transduction events and the role of immune mechanisms in alcoholic liver disease have been some of the key areas of investigation. Our goal is to translate to humans how factors in the exposome such as age, nutritional status, cigarette smoking, and exposure to pathogens interact in the presence or absence of alcohol administration. We know that a major component of the specific external environment is the lifestyle factor of alcohol consumption. Thus, under the comprehensive schema of the exposome, alcohol represents an environmental exposure that should be considered in context with other internal and external factors to understand causes and nature of disease progression. The overall hypothesis of our application is the following: the examination of alcohol in the context of the exposome will allow us to understand its role more fully in the etiology of disease and the subsequent manifestations of alcohol-induced human pathophysiology. The alcohol-exposome theme of the center is unique among alcohol centers and has the potential to be highly translational. Our investigators represent an outstanding team of interdisciplinary investigators that will be supported by an Administrative Core, a Biospecimen Core, and a Pilot Projects Core. ACORN investigators will have access to state-of-the art facilities at UNMC, Nebraska Medical Center and University of Nebraska-Lincoln. We include faculty from 5 departments and 3 colleges. Our proposed Specific Aims for the ACORN are to: 1) Facilitate interdisciplinary research and identify factors so that we can translate in humans how age, nutritional status, cigarette smoking, and exposure to pathogens interact in the presence or absence of alcohol administration. 2) Create a Biospecimen Core to aid Center investigators in promotion and development of translational animal and human tissue and cell models. 3) Enhance the collaborative ACORN environment and provide opportunities for funding through training, symposia, and pilot grants. Work on our 4 research projects, with participation of the Administration Core and the Biospecimen Core will allow us to realize our goals. Overall, this new P50 will utilize the expertise and knowledge of past research efforts to examine how alcohol, in the setting of various external and internal environmental factors will influence disease in a variety of organs and help us shape our future animal and human studies.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Fracture is the leading musculoskeletal injury affecting over 9.4 million Americans annually. Approximately ~10% of fractures are complicated by delayed healing or non-union, resulting chronic pain, impaired mobility and poor quality of life. Pathophysiological and habitual risk factors, including fracture site instability, concurrent diseases (e.g., diabetes), chronic exposure to certain medications (e.g., glucocorticoids or GCs) and tobacco/alcohol use are also major contributing causes, which may interfere with the fracture healing biology and lead to delayed healing or non-union. GCs are widely used in treating hospitalized COVID patients. Given the scale of the current pandemic and the significant COVID infection rate among the US population, a post pandemic surge of fracture risk and delayed fracture healing cases may be anticipated. Currently, orthopaedic surgery and autologous bone grafts are still the gold standard in clinical management of delayed fracture healing. There are few FDA approved non-invasive therapeutic interventions for the treatment of GC-induced delayed fracture healing. To address this significant unmet clinical need, we have developed a N-(2- hydroxypropyl)methacrylamide (HPMA)-based water-soluble thermoresponsive polymeric prodrug (P-TAN) of Tanshinone IIA (TAN, a potent bone anabolic agent). The aqueous solution of P-TAN is free-flowing at room temperature and transitions into a hydrogel (ProGel-TAN) at ≥ 27°C, which provide a unique mechanism for sustained local delivery of TAN. When tested in a prednisone-induced delayed fracture healing mouse model, two consecutive monthly ProGel-TAN treatments were found to accelerate the fracture healing by 5 weeks without any side effects observed. The biomechanical properties of the bone were also fully restored. While two monthly ProGel-TAN treatment seems to be very promising, the current orthopaedic practice favors a single dose treatment at the time of the fracture stabilization procedure for pragmatic reasons as well as less infection risk. Therefore, we hypothesize that with further structural and formulation optimization, we will be able to identify an optimized single dose ProGel-TAN formulation that is highly effective and safe in treating the GC-induced delayed fracture union. To test this hypothesis, we propose three Specific Aims in this project: 1. To characterize the impact of different structural and formulation parameters of ProGel-TAN; 2. To identify single dose ProGel- TAN formulations that provide optimal fracture healing efficacy; 3. To understand ProGel-TAN’s unique pharmacology and putative mechanisms of action. At the successful completion of the proposed research, we anticipate the identification of at least one optimal ProGel-TAN formulation that would satisfy the demand for a single dose treatment to accelerate the healing of the GC-induced delayed fracture union by ~ 5 weeks and normalize biomechanical properties of the injured bone in mice. It will then be subjected to further pre-clinical studies on large animal models in preparation for clinical translation.

NIH Research Projects · FY 2025 · 2022-09

Abstract Claudication, the most common clinical presentation of patients with Peripheral Artery Disease (PAD), is a severe functional limitation of the lower extremities identified as walking-induced leg muscle pain relieved by rest. Numerous studies have identified a lower-leg myopathy in these patients. There is general agreement that the proximate cause of this myopathy is dysfunctional mitochondria which produce oxidative damage in response to repeated episodes of walking-induced ischemia/hypoxia. These patients have few therapeutic options including only two FDA approved medications, Pentoxifylline and Cilostazol, which are modestly effective. A promising medication for treatment of claudicating PAD patients is MitoQ an antioxidant that concentrates several hundred-fold in mitochondria. The significant contribution of mitochondrial oxidative damage to a wide range of pathologic conditions has stimulated clinical studies which have found MitoQ to be safe and effective. Our group has documented improved walking performance of claudicating PAD patients receiving a single dose of MitoQ. We propose to study, for the first time, the effects of long-term MitoQ treatment on the myopathy and functional performance of claudicating PAD patients. Our Hypothesis: Treatment of claudicating PAD patients with MitoQ for six months improves 1) patient performance determined as walking performance, daily physical activity, and quality of life, 2) calf muscle histopathology and pathophysiology, and 3) the systemic physiological parameters pulmonary O2 uptake (VO2) and metabolic profile. These changes correlate directly with reduced oxidative damage to calf muscle mitochondria, improved mitophagy, and improved mitochondrial function. We will test this hypothesis by implementing the following Specific Aims. Specific Aim ‘1’ will test the hypothesis that a six-month regimen of MitoQ improves performance determined as walking performance, daily physical activity, and quality of life of claudicating PAD patients, in association with improved calf muscle histopathology & pathophysiology, and improved VO2 & systemic metabolic profile. Specific Aim ‘2’ will test the hypothesis that a six-month regimen of MitoQ reduces mitochondrial oxidative damage, improves mitophagy, and improves mitochondrial function of the voluminous, myofiber-mitochondrial compartment and that these improvements correlate with improved performance of claudicating PAD patients, improved calf muscle histopathology & pathophysiology, and improved VO2 & systemic metabolic profile. Specific Aim ‘3’ will test the hypothesis that a six-month regimen of MitoQ improves endothelial function and lower extremity hemodynamics, calf muscle heme oxygenation, and endothelium-dependent vasomotor function of micro-vessels isolated from the affected calf muscle of claudicating PAD patients, in association with improved mitochondrial function of the micro-vessels. If our hypothesis is correct, the work will support a causal connection between mitochondrial oxidative damage and PAD myopathy and patient performance; and identify MitoQ as a promising treatment for PAD.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Musculoskeletal disorders are among the most common causes of acute and chronic pain. Extensive use of opioids for these conditions has made a substantial contribution to the current opioid epidemic. There is an urgent need to develop potent analgesics that are as effective as opioids but avoids the side effects, including the risk of dependency and addiction. To address this challenge, we have developed a macromolecular prodrug (P-HMP) of hydromorphone (HMP) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. When HMP content increased to ³ 16 wt%, the aqueous solution of P-HMP becomes thermoresponsive and transitions from a liquid at 4°C to a hydrogel at ³ 30°C. The hydrogel, which was designate as ProGel-HMP, allows the sustained retention of the opioid prodrug at sites of pathology. When tested in the destabilization of the medial meniscus (DMM) model of osteoarthritis (OA), ProGel-HMP provided potent and sustained (³ 16 days) pain relief without spinal cord analgesia. Once locally administration, P-HMP slowly dissolves from ProGel-HMP and is processed subcellularly by phagocytic cells to release HMP for sustained local pain relief. The P-HMP that is not sequestered by cells drains into the circulation and rapidly clears via the kidney, effectively limiting systemic exposure. Importantly, the absence of spinal cord analgesia, indicates that the thermoresponsive P-HMP does not permeate the blood-brain or spinal cord barriers, which circumvents the risks of eliciting centrally mediated drug-dependency and addiction associated with conventional opioids. Based on these preliminary findings, we hypothesize that (1) that ProGel-HMP can provide sustained and effective local post-operative analgesia and pain amelioration at sites of musculoskeletal trauma, independent of CNS-mediated effects; (2) that the formulation parameters of ProGel-HMP can be modified to regulate the duration and efficacy of local analgesia to meet the specific needs for pain management in different musculoskeletal conditions. To test these hypotheses, we propose to first establish the dimensions within which the ProGel-HMP formulation parameters can be adjusted to regulate the duration and efficacy of local analgesia (Aim 1). Three pain-causing conditions will then be simulated in mice to allow in vivo assessment of ProGel-HMP. The DMM mice will be used to identify optimal ProGel-HMP formulations for both short-term amelioration of post-operative pain and sustained amelioration of chronic OA pain. In addition to testing efficacy and safety, pharmacokinetics/biodistribution (PK/BD) and functional physiological studies will be performed to dissect ProGel-HMP’s mechanism of action (Aim 2). The final studies will employ a murine closed fracture model to screen for an optimal ProGel-HMP formulation that provides medium-term pain relief in skeletal trauma through the stages of tissue inflammation and early skeletal repair. Efficacy, safety and mechanistic studies that are similar to those in Aim 2 will be performed (Aim 3). At the successful completion of this proposal, we will amass robust evidence supporting our central hypotheses that will ultimately lead to the successful translation of ProGel-HMP into clinical practice.

NIH Research Projects · FY 2025 · 2022-09

Abstract: An estimated ~1.3 million adults and adolescents are living with human immunodeficiency virus (HIV) infection in the USA of which 15% of them undiagnosed. The initiation of combination antiretroviral therapy (cART) has transformed AIDS from a fatal illness into a chronic and manageable disease. The presence of HIV infected microglia and macrophage reservoirs in the central nervous system (CNS) and variable penetration of anti- retroviral drugs across the blood brain barrier after cART are likely factors for the persistence of HIV associated neurocognitive disorder (HAND). Drugs of abuse such as the potent psychostimulant methamphetamine (Meth) that is abused by an estimated 30 million people in the world further minimize the efficacy of cART and in conjunction with HIV exacerbate CNS pathology. Thus, significant gap in knowledge in the field is to better understand the neuropathogenesis and the etiology of clinical outcomes observed in HIV+ patient’s dependent on Meth abuse. The current grant proposal focuses on developing a human induced pluripotent stem cell based cerebral organoid model to investigate underlying brain dysfunction during HIV/Meth interaction. Cerebral organoids are 3-dimensional “mini brains” that can self-organize and recapitulate many milestone events seen in in vivo brain development. Our recent study on generating a novel organoid model with the feasibility in controlling the microglia ratio and microenvironment of organoid formation to recapitulate changes in brain functions under various conditions (e.g. virus and viral infection-induced inflammation and synaptic pruning) makes this model ideal for our proposed studies. The central hypothesis of this proposal is that Meth in conjunction with HIV causes significant neuronal damage. The combination of microglia-containing cerebral organoid model, extracellular vesicle biology, single-cell RNA-sequencing, CRISPR/Cas9-mediated gene editing will decipher novel intercellular and molecular mechanisms and pathways that underlie abnormalities in neuronal functions and connectivity caused by HIV infection. We will investigate this hypothesis under three specific aims: Specific Aim 1 will examine neuronal function, synaptic and mitochondrial perturbations during HIV/meth interactions in 3D microglia-containing cerebral organoid model; Specific Aim 2 will investigate the role of extracellular vesicles in microglial organoids treated under HIV/meth conditions; and Specific Aim 3 will characterize the molecular mechanisms underlying HIV infection-induced neuronal injury and further evaluate our microglial organoid model as a reliable tool to identify molecular signatures of HAND. Our proposed experiments will decipher molecular mechanisms, novel signaling events and molecular partners underlying the neuronal injury in HAND. Through the series of the experiments we aim to present an easily scalable and reproducible model to study HAND pathogenesis. The data obtained from these studies can be used to design novel therapeutics to control HIV infection.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY/ABSTRACT The Central States Center for Agricultural Safety and Health (CS-CASH) will conduct high quality research and translate scientific discoveries into practical applications to reduce the burden of injury and illness among farmers, ranchers, workers, and their families. The Center has high scientific and technical competency, and it is forward looking. CS-CASH with a strong network of collaborators provides regional leadership in research and outreach. The Center takes advantage of the University of Nebraska Medical Center's (UNMC) strengths in public health research and strong institutional support. Research teams from several states and institutions bring multi-disciplinary expertise and access to special populations. With our advisors and partners, we can leverage the Center's resources to address local, regional, and national issues. CS-CASH has built a cohesive approach that links evaluation, planning, research, and outreach to reduce agricultural occupational injury and illness. The Center's Evaluation and Planning Core provides strategic direction, administration, and evaluation for the Center, and it responds to emerging issues. The Research Core conducts basic, intervention, translation, and surveillance research, and manages pilot/feasibility projects with substantial in-kind support from UNMC. The research themes focus on safety and health in row crop and livestock production, and surveillance of injuries, illnesses, exposures, and related costs. The specific projects titles are: Exposome and organic dust-induced lung injury; Safety of agricultural machinery ingress/egress for aging producers; Impact of stress among Latino immigrant workers; Respiratory health in poultry production: exposures, health effects, and options for protection; Community-based training network to enhance bison worker safety on tribal lands; Safety climate and culture in the cattle feedyard industry; and Surveillance of agricultural injury, illness, and stress in the Central States region. The Outreach Core uses a multi-faceted approach in reaching out to target populations, promoting the Center's themes in `boots-on-the-ground' events, and communication through traditional and social media. The Center has a special emphasis on vulnerable populations: women, young farmers, aging farmers, veteran farmers, Latino immigrant workers, and Native American workers. CS-CASH partners with neighboring Centers to identify synergies, utilize our collective strengths, and avoid duplication of efforts. CS-CASH is well-established with a clear vision, mission, strategy, organization, and service area. With the new proposed lines of research and outreach, the Center is well positioned to serve the Center's seven-state region and make an impact in reducing agricultural injury and illness during the 2022-2027 funding cycle.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The human genome is precisely folded into various structures, which provides the context for functional processes in the nucleus. Recent advances have indicated the presence of chromatin compartments, CTCF loops, and small gene-loops as prevalent features of genome organization. Our investigations at high resolution reveal the presence of sub-genic discordant compartments, placing the 5’ and 3’ ends of many genes in opposite compartment states. These, along with gene-loops, are highly correlated with transcription elongation status, but how transcription influences these sub-genic structures is unknown. Additionally, the spatial location of these features is an important aspect of genome organization that is largely ignored, and which of these features are dependent on radial location is unclear. The goals of our research are to determine the relationship between transcription and sub-genic scale chromatin organization and to elucidate the impact of spatial location on chromatin folding. We will accomplish these goals in programs that use (1) basic mechanistic interrogation to track how sub-genic structures change as transcription progresses, (2) software development for increased sensitivity and quantitative fidelity in fine-scale compartment analysis, and (3) investigative genomics to enrich for peripheral interactions and define the relationship between chromatin contacts and radial position. The results of this research will resolve mechanistic questions surrounding transcription and gene folding, provide new sorely needed and cost-saving tools for fine-scale analysis, and identify spatially dependent chromatin organization.

NIH Research Projects · FY 2025 · 2022-09

Metastatic castration-resistant prostate cancer (mCRPC) is deadly and currently incurable. Approximately 90% of patients CRPC become resistant to 2nd line androgen deprivation therapy (ADT; which primarily target androgen receptor (AR) signaling and present with bone metastatic disease. Although ADT remains a beneficial therapy for mCRPC patients, mechanisms of cancer resistance in mCRPC and specifically, in the bone environment, the most frequent site of CRPC metastasis, is poorly understood. Understanding contributing factors to PCa disease progression is needed for further development of efficacious therapies. ADT was previously shown to be critical for differentiation and function of polymorphonuclear leukocytes/neutrophils (PMNs) which are “first responder” innate immune cells that comprise ~40-50% of the bone marrow cavity. We recently showed that PMNs are protective against bone metastatic prostate cancer (BM-PCa) however, the PMN anti-tumoral immune response diminishes as the tumor progresses. To examine PMN phenotypical changes throughout PCa progression in patients, my group functionally and molecularly characterized peripheral blood PMNs from PCa patients at different stages: 1) Localized PCa, 2) bone metastatic hormone-sensitive (mCSPC), and 3) mCRPC patient. We found that PMN function was highly suppressed by 2nd line ADT through increased receptor 1 expression of transforming growth factor beta (TGFβ), an anti-inflammatory cytokine important for promoting BM-PCa and cancer-induced bone disease. Using preclinical bone metastasis mouse models, we were able to significantly suppress mCRPC growth in bone using 2nd line ADT in combination with either bipolar androgen therapy (BAT; exogenous testosterone) to boost PMN anti-tumor response OR PMN-specific genetic deletion of TβR1. Based on our preliminary findings, we hypothesize that: anti-tumor PMNs are suppressed/ “switched off” by androgen regulation via TβR1 signaling and this can be leveraged to improve mCRPC outcomes. This will be tested in the following aims: Aim 1. Define the impact of androgen regulation on PMN anti-tumor immune response. Aim 2. Determine the mechanism of TβR1-mediated PMN immune response in BM-PCa. Aim 3. Delineate the therapeutic potential of dual TβR1/AR regulation for improving mCRPC therapeutic outcomes. Primary Objective: To develop a novel immunotherapeutic strategy for treating BM-PCa by enhancing PMN anti-tumor response and overcoming PCa resistance to ADT. Study Design: For Aim 1, we will identify the impact of androgen signaling on PMN polarization ex vivo (using patient-derived PMNs and mouse bone marrow PMNs) and in vivo using normal PCa, non-metastatic and bone metastatic PCa cells) and in vivo (using mouse intratibial bone metastasis models). For Aim 2, we will delineate the role of TβR1 in PMN response to mCRPC using TβR1 knockout models. For Aim 3, we will define the therapeutic potential for using combination BAT with a novel bone-targeted TβR1 inhibitor.