University Of Nebraska Medical Center

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY The goal of this study is to elucidate novel mechanisms underlying exercise training (ExT)-induced sympatho- inhibition in chronic heart failure (CHF) (Aim 1) and cardioprotection following acute coronary ischemia/reperfusion (I/R) (Aim 2), based on an innovative concept of inter-organ transfer of antioxidant enzymes. It is well established that exercise generates muscle-derived reactive oxygen species (ROS) which activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), resulting in upregulation of a panel of antioxidant enzymes in skeletal muscle per se. We hypothesize that these antioxidants can be transported from skeletal muscle to remote tissues through circulating extracellular vesicles (EVs), providing recipient cells with a second and enhanced line of antioxidant defense. In the rostral ventrolateral medulla (RVLM) of mice with CHF, these antioxidants restore redox homeostasis of pre-sympathetic neurons, contributing to ExT-sympatho- inhibition. Furthermore, in the heart of mice subjected to coronary I/R, these antioxidants reduce free radical damage and salvage ischemic myocardium, thus playing a critical role in ExT-cardioprotection. To address these hypotheses, we developed three skeletal muscle-specific transgenic mouse lines. The MS-mG line is a reporter model, which allows us to track, capture, and analyze EVs released specifically from skeletal muscle. This model will be used to assay cargo proteins of skeletal muscle-derived EVs and their distribution in brain and heart following ExT. The iMS-Nrf2flox/flox and iMS-Keap1flox/flox lines will enable us to delete skeletal muscle Nrf2 (i.e. Nrf2 deficiency) and Keap1 (i.e. Nrf2 overexpression), respectively. These two models will be used to demonstrate a causal relationship between the Nrf2/antioxidant system and ExT-sympatho-inhibition in Aim 1 and ExT-cardioprotection in Aim 2. Interdisciplinary methods of EV biology, proteomics, bioinformatics, electrophysiology, and cardiovascular physiology will be utilized to characterize skeletal muscle-derived EVs following ExT, determine the effects of ExT-EVs on central neuron discharge and peripheral sympathetic nerve activity, and explore the mechanisms underpinning cardioprotection of ExT-EVs against coronary I/R injury. Upon completion of this project, we expect to provide novel mechanistic insights on ExT-cardiovascular protection, paving a new avenue to translate the beneficial effects of regular physical activity into clinical practice to prevent and treat acute and chronic ischemic heart diseases.

NIH Research Projects · FY 2026 · 2022-01

Abstract/Project Summary The United States population is becoming increasingly older and the prevalence of Alzheimer’s disease (AD) and its common precursor, mild cognitive impairment (MCI), is expected to dramatically rise in the coming years. As such, there is an immediate national need to further understand the neurophysiological basis of these neuro- degenerative diseases. Recent studies have shown disruption of gamma-band neural oscillatory activity in animal models of AD. Further, visual stimulation at gamma frequencies has been shown to increase the clearance of amyloid-β (Aβ) and hyperphosphorylated tau in mouse models, while improving cognitive performance. Despite these groundbreaking findings, research into gamma oscillatory activity in humans with AD remains scarce. Relatedly, visuospatial attention processes are among the earliest and most severely affected cognitive functions in MCI and AD. Dysfunction in this domain has also been shown to be more specific for identifying Alzheimer’s type dementia relative to other forms (e.g., frontotemporal dementia) when compared to dysfunction in other cognitive domains (e.g., memory). Although neuropsychological testing has shown clear deficits in visuospatial attention in patients with MCI and AD, very little is known about the neural oscillatory activity that underlies these deficits. The current study aims to partially remedy these knowledge gaps by utilizing the spatial precision and exquisite temporal resolution (i.e., millisecond) of magnetoencephalographic (MEG) imaging. Briefly, persons with AD, MCI, and demographically-matched controls will complete two cognitive tasks during MEG recording; one examining multispectral visual entrainment activity and another investigating visuospatial attention processing. The resulting MEG data will be transformed into the time-frequency domain and imaged using a beamforming approach. The output dynamic functional maps of electrical neural activity will be used to examine baseline and task-related entrainment and oscillatory activity among regions serving visual and visuospatial processing. In particular, we will identify the statistically anomalous neural oscillations in patients with MCI and AD, and then link these neural data to regional Aβ deposition and overall performance (e.g., general and domain-specific cognition, functional capacity, etc.). Our Aims are: (1) To quantify the cortical dynamics during visual entrainment at specific frequencies (i.e., 20, 35, 40, and 45 Hz) in patients with MCI or mild AD, and decifer the relationship between local gamma activity and quantitative Aβ deposition, and (2) to identify deficits in the tracking of attended visual stimuli in patients with MCI and mild AD, and determine how this relates to cognitive performance. To this end, we will utilize the latest MEG and source reconstruction techniques, neural oscillatory analysis methods, quantitative Aβ PET imaging, and neuropsychological assessments to delineate the neurophysiological bases of cognitive impairments in patients with MCI and AD. This research will aid in illuminating the neural dynamics underlying cognitive dysfunction in those with MCI and AD, with the primary goals of scientific discovery and developing the research and clinical skills of the applicant to produce a successful physician-scientist.

NIH Research Projects · FY 2025 · 2022-01

Abstract Abdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta with a high mortality greater than 80% after rupture. Aortic vascular smooth muscle cells (SMCs) are pivotal in maintaining aortic structural integrity and function, and SMC-rich aortic medial stability is highly disrupted in AAA. Currently, besides surgical interventions, no alternative therapeutics are available to blunt AAA progression and rupture. Consequently, there is a dire need to identify novel strategies for development of effective, non-surgical therapeutics. MicroRNA-146a (miR-146a), a well-known regulator of inflammation and auto-immunity, is highly expressed in aneurysmal tissue of AAA patients. However, the role of miR-146a in SMC homeostasis and AAA remains to be explored. In preliminary studies, by in-situ hybridization, we observed that miR-146a is upregulated in SMC-rich aortic media of human and mouse AAAs; miR-146a deficiency significantly promoted Angiotensin II (AngII) -induced AAA formation in normolipidemic mice co-administered with Lysyl oxidase inhibitor, β-aminopropionitrile (BAPN); and mimetics-mediated miR-146a overexpression abolished AngII-induced AAAs in both hypercholesterolemic LDLr-/- mice and normolipidemic mice co-infused with BAPN. To elucidate underlying mechanisms, by RNA sequencing, we identified novel targets from miR-146a deficiency experiments: TFIID-31, a TATA binding protein associated factor involved in transcriptional activation and repression, is significantly upregulated; whereas Beclin-1, a gene indispensable for autophagy induction and USP9X, a deubiquitinase critical for Beclin-1 stabilization, are significantly downregulated. Autophagy, a self-regulatory process by which cells digest, and recycle their cytoplasmic materials for energy purposes under stress. Our preliminary study also showed an increased Beclin-1 in mouse AAAs, as observed in human AAAs and Tat-peptide mediated Beclin-1 activation suppressed AngII-induced AAA formation in mice. In addition, miR-146a overexpression significantly suppressed TFIID-31, promoted USP9X and Beclin-1, and ShRNA-mediated silencing of TFIID-31 increased USP9X in cultured aortic SMCs. Based on these observations, we will test our central hypothesis that miR-146a activation protects against AAA formation and progression by promoting Beclin-1-mediated aortic SMC homeostasis. By utilizing our unique mice models generated specifically for these studies, we propose 3 aims. Aim 1 will test our working sub-hypothesis that miR-146a promotes Beclin-1 stability in aortic SMCs via a TFIID-31-USP9X –dependent manner. Aim 2 will test our working sub-hypothesis that miR-146a activation protects against AAA through activation of SMC-Beclin-1-derived autophagy. Aim 3 will determine the effect of miR-146a / Beclin-1 activation on progression of established AAAs. In summary, we will delineate the protective role of Beclin-1 in AAA and establish miR-146a activation as a novel therapeutic strategy against AAA by targeting SMC-Beclin-1. This mechanistic research will set solid preclinical evidence that targeting miR-146a represents a novel therapeutic strategy for treatment and prevention of AAA.

NIH Research Projects · FY 2025 · 2021-12

Community acquired pneumonia (CAP) is a leading cause of morbidity and mortality worldwide. Streptococcus pneumoniae (pneumococcus) remains the most common cause of CAP in the U.S. The incidence of CAP continues to rise contributing to increased hospitalization and mortality. A major cause of CAP is decline in immune function in vulnerable populations. Zinc (Zn) is required for proper immune function and insufficient dietary intake is highly prevalent within vulnerable populations. Zn deficient subjects are more susceptible to pathogens and have a higher incidence of pneumonia whereas Zn supplementation reduces the incidence of pneumonia. The long-term goal of this project is to determine the role of the human zinc transporter ZIP8 and dietary Zn intake in the host immune response to pneumococcal pneumonia. Our group was the first to reveal that ZIP8 is required for myeloid cell activation following exposure to bacteria. This is relevant because a relative deficit of Zn, either by dietary restriction or deficits in ZIP8-mediated Zn transport, in the setting of the host response to bacterial invasion in the lung leads to immune dysfunction, increased lung damage, and higher mortality (see preliminary data). We hypothesize that ZIP8 plays a pivotal role in lung macrophages and dendritic cells by maintaining favorable balance of both the innate and adaptive immune response. Accordingly, defective Zn intake or ZIP8 function prohibits the ability of Zn to facilitate normal immune function and host defense. If proven correct, this will have important implications on pneumonia pathogenesis and increase our capacity to predict disease susceptibility and prevent morbidity and mortality. Guided by strong preliminary evidence, this hypothesis will be tested by pursuing three specific aims that will: 1) Determine the impact of ZIP8 loss on the lung myeloid landscape in vivo and its impact on pathogen clearance and host survival; 2) Determine how ZIP8 impacts Macrophage and DC function; and 3) Determine the impact of Zn supplementation on pneumococcal pneumonia in vivo in the setting of Zn dyshomeostasis. To accomplish our goals we have assembled a strong and experienced team that will pursue novel studies in two novel knockout mouse models and a model of dietary Zn restriction that will explore the role of the zinc transporter protein ZIP8 in maintaining myeloid cell-driven immune balance in the setting pneumococcal pneumonia. At the successful completion of this study, we will better understand the interplay between Zn homeostasis and ZIP8 in the context of pneumococcal infection in the lung. This is expected to have a positive impact because it will reveal previously unidentified molecular pathways that are instrumental in host defense. Further, we have the potential to identify novel micronutrient and genetic surveillance as well as treatment strategies that will improve our ability to prevent pneumococcal pneumonia in the most vulnerable populations worldwide. We envision that this will also lend itself to new approaches to treat or prevent other harmful pathogens.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Cancer and treatment-related cognitive changes cause distress, hinder resumption of normal routine and roles, and worsen quality of life. Hematopoietic cell transplantation (HCT) is a potentially life-prolonging treatment for patients with hematological malignancies. HCT differs from the delivery of chemotherapy in other cancer settings due to the intensity of chemotherapy and severity of toxicity. Previously reserved for younger patients, in the past decade a growing number of adults 60 years of age and older have received this intense treatment. Our preliminary data shows that post-HCT, 35% of older patients experience significant cognitive impairment compared to 19% pre-HCT. Physical activity improves cognitive function in older adults and survivors of other cancers. While untested in older adults undergoing HCT, we hypothesize that increasing physical activity can also improve cognitive function in this vulnerable population. The objective of this proposal is to adapt and test an evidence-based physical activity intervention, The Community Health Activities Model Program for Seniors II (CHAMPS II), in the HCT setting to improve cognitive function for adults 60 years and older. This program is effective in increasing physical activity for sedentary community-dwelling older adults with multiple chronic health conditions. We will iteratively adapt and implement CHAMPS-II in three waves. During each wave, 2-4 patient/care-partner dyads will participate in a multi-phase, light- to moderate-intensity physical activity program. We will use a Type 1 hybrid effectiveness-implementation design to determine the preliminary effectiveness of the adapted intervention to improve cognitive function compared to an active control condition (N = 34 per group). This novel trial design spans the effectiveness and implementation research to accelerate the translation of physical activity interventions into practice for older adults with cancer. The translation of physical activity interventions into clinical practice remains challenging within cancer survivorship, despite promising evidence of efficacy in improving functional outcomes and quality of life. To become an independent researcher who develops, implements, and disseminates interventions that prevent cognitive decline and improve functional outcomes for older patients with cancer, Dr. Koll requires additional training and experience in: 1) physical activity promotion and principles of exercise training; 2) design and conduct of hybrid effectiveness-implementation clinical trials; and 3) leadership and project management skills needed to execute translational studies requiring collaboration between researchers and clinicians. An outstanding team of mentors and advisors support this proposal with expertise in geriatrics (Fisher), behavioral and implementation science (Estabrooks), clinical exercise trials in older adult and cancer populations (Ehlers), geriatric oncology (Wildes), cognitive disorders and cognitive aging (Murman), and mixed methods (Jones). The training and research plan will position Dr. Koll to become one of the few geriatricians with expertise in cognition and cognition-enhancing interventions in older adults with cancer.

NIH Research Projects · FY 2025 · 2021-09

Alcohol use disorders (AUDs) are common, disabling conditions. There is a growing awareness of important sex differences in AUDs; for example, women experience more serious health complications from alcohol use and also develop negative consequences from alcohol use more quickly. While the rate of AUDs is relatively stable in men, the rate in women is escalating at an alarming rate. Neurobiological differences between sexes are thought to underlie the differential impact of AUDs in men and women, but to date relatively few studies on this topic exist. Animal models of addiction have substantially informed our understanding of the stages of addiction— binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation—and their underlying pathophysiology. For example, chronic alcohol exposure causes neuroadaptive brain changes in an attempt to maintain homeostasis. During the withdrawal/negative affect stage, hyperactive stress systems produce symptoms including anxiety and depression which are thought to lead to relapse through negative reinforcement. Women have higher rates of anxiety and stress-related disorders, which may contribute to sex differences in early abstinence. Animal models of early abstinence from alcohol highlight the involvement the bed nucleus of the stria terminalis (BNST). The BNST is also one of the most sexually dimorphic brain regions, suggesting the BNST is involved in sex-related differences seen during early abstinence. We previously published evidence for sex differences in BNST structural connectivity in humans. In addition, pilot data from our NIAAA funded R21 provide initial evidence for sex differences: females had stronger structural and functional connectivity within the BNST network, heightened stress responses, and higher anxiety during early abstinence. The current study will focus on sex differences in the BNST network during early abstinence from alcohol by investigating three specific aims: (1) Determine whether there are sex-related differences in BNST intrinsic functional or structural connectivity during early abstinence; (2) Determine whether there are sex- related differences in stress-related BNST function and BNST network connectivity during early abstinence; (3) Investigate sex-related differences in the relationship between BNST function/connectivity, stress response, and negative affect in early abstinence. Based on findings from animal models and our pilot data in humans, we predict that during early abstinence, the BNST will show sex-specific differences in patterns of activity and connectivity “at-rest” and in response to a mildly stressful task. We expect women will show stronger structural and functional connectivity within the BNST network, heightened stress responses, and higher anxiety during early abstinence. The successful completion of this study will fill a critical knowledge gap, determining whether men and women show neurobiological differences in BNST networks underlying negative affect during early abstinence from alcohol. The results will provide foundational information to inform future studies investigating mechanisms of relapse and can guide the development of sex-specific or personalized treatments for AUD.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT CLN3 and CLN6 disease are subtypes of a wider family of pediatric neurodegenerative diseases called the Neuronal Ceroid Lipofuscinoses (NCLs) or Batten Disease. The NCLs affect approximately 6-8 children per 100,000 live births worldwide. Common symptoms of CLN3 and CLN6 disease include vision impairment which progresses to blindness, seizures which increase in severity, cognitive and motor decline progressing to dementia, and ultimately premature death. No cure or effective treatment for either CLN3 or CLN6 disease is known. The development of new disease-modifying agents to treat CLN3 and CLN6 disease is an urgent and unmet medical need. Common phenotypes that are shared between all NCLs include dysfunctional autophagy leading to accumulation of storage material, reduced expression of the anti-apoptotic protein Bcl-2 and increased ceramide production leading to apoptotic death of neurons, and dysfunctional mitochondria. Autophagy and apoptosis are physiological process that contribute to cellular homeostasis. Dysfunction of one, or in many cases, both processes, is phenotypic across many neurodegenerative diseases in addition to the NCLs. While targeting either process individually results in promising pharmacological effect, no small molecule has been identified that is capable of modulating both synergistically. While a multi-target approach has been used in cancer treatment for many years, it has only recently begun being applied to neurodegenerative diseases and has yet to be explored in CLN3 and CLN6 disease. Through a structure-based approach, we have identified a library of multi-functional compounds that fuse autophagy activation activity, anti-apoptotic Bcl-2 induction and decreased ceramide synthesis resulting in translational activity to protect human induced pluripotent stem cell (iPSC)-derived neurons from externally- induced and phenotype-induced apoptosis. Moreover, we have shown in our preliminary data that iPSCs obtained from CLN3 patients and derived to functional neurons recapitulate the aberrant autophagy, apoptosis and mitochondrial function phenotype of the disease, and that these phenotypes can be rescued by selected lead compounds. Further, we show proof-of-concept that our lead compounds rescue CLN3 disease phenotypic behavioral deficits in a transgenic CLN3 mouse model. The goal of this application is to optimize our proprietary library of neuroprotective compounds to further understand their minimum pharmacophore, confer ‘drug-like’ properties, identify and eliminate any potential toxicity, optimize drug metabolism and pharmacokinetic parameters, further credential their mechanism of action and demonstrate proof-of-concept protective activity in additional patient iPSC-derived neuron lines and CLN3 and CLN6 transgenic mice.

NIH Research Projects · FY 2025 · 2021-09

Abstract Peripheral T-cell lymphomas (PTCL) represent approximately 12-15% of all NHL in the western world and are associated with dismal prognosis. Furthermore, the diagnosis is challenging as 30-50% of PTCL cases cannot be assigned to a specific entity and are categorized as PTCL-not otherwise specified (PTCL-NOS). We have defined robust gene expression signatures that can differentiate the five common PTCLs entities: angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase positive anaplastic large-cell lymphoma (ALK (+) ALCL), ALK- negative anaplastic large-cell lymphoma (ALK (-) ALCL), adult T-cell leukemia/lymphoma (ATLL), and extra-nodal natural killer/T-cell lymphoma (ENKTCL). PTCL-NOS can be divided into two distinct biological and prognostic subgroups (PTCL-TBX21 and PTCL-GATA3 subgroups). We translated the RNA based diagnostic and prognostic algorithms for formalin fixed paraffin embedded (FFPE) tissues for widespread clinical usage with high sensitivity and specificity. We also identified distinguishing genetic lesions in PTCL subtypes using corresponding DNA , and demonstrated that such lesion can be validated using shallow whole genome analysis (sWGA) in corresponding plasma cell-free DNA, thus liquid biopsy can aid in diagnosis and disease monitoring. Since the biospecimen processing, and hence quality, varies significantly in routine clinical pathology laboratories, the reliability of RNA or DNA based signatures need to be evaluated under variable circumstances. It is essential to determine how the robustness of the assay may be affected by pre-analytical variables before the novel diagnostic tools can be applied to large studies or routine clinical practice. We hypothesize that a comprehensive evaluation of pre-analytical variables of biospecimen will lead to optimized bio-specimen procurement framework leading to improved diagnostic accuracy and reproducibility in tissue and liquid biopsy setting and can be standardized in an inter-CLIA lab setting for routine clinical practice/trials. This proposal aims to establish standardized, evidence-based procedures on bio-specimen (RNA/DNA) processing, storage and transportation to ensure accurate, reproducible assay performance. The identified conditions and parameters will be validated on prospective samples, preferably in a clinical trial setting, so findings can be correlated with clinical data. Thus, three specific aims are proposed: Specific Aim 1: To determine pre-analytical variables that affects the reliability of RNA-based assays in FFPE tissue Specific Aim 2: To identify pre-analytical factors affecting circulating tumor DNA (ct-DNA) detection and quantification in patients with PTCL Specific Aim 3: To validate harmonization of the pre-analytical variables in improving PTCL diagnostic or prognostic assay in an inter-CLIA (Clinical Laboratory Improvement Amendments) lab setting The studies will lead to robust protocols that optimize the preservation biomolecules in tissue biopsies or plasma to ensure accuracy and reproducibility of molecular assays, improving PTCL classification and prognostication.

NIH Research Projects · FY 2024 · 2021-09

Abstract Pancreatic cancer (PC) is one of the most lethal cancers in the U.S. with a 5-year survival rate of 7%, which has essentially unchanged for the past 40 years. Despite extensive research, there are no effective targeted therapies for this disease, suggesting an insufficient understanding of oncogenic signaling networks in PC. While almost all of the signaling networks implicated in cancer rely on the cooperation of kinases and phosphatases to transduce signals promoting cancer, most efforts have only been put on the study of protein kinases in cancer while leaving protein phosphatases as a blind spot. PP2A is a family of heterotrimers that serve as the main source of Ser/Thr phosphatase activities in human cells, with each consisting of a scaffold subunit (Aα/β), a catalytic subunit (Cα/β), and one of many non-conserved regulatory (B) subunits (>27) that control the substrate specificities of PP2A complexes. Our preliminary studies identified PR55α, a regulatory subunit of PP2A, as an important new player in promoting malignant PC. In particular, our studies revealed that PR55 inhibits the MOB1/LATS auto-activation loop of the Hippo tumor suppression pathway, whose function is to induce the phosphorylation of YAP causing its cytoplasmic retention and proteasomal degradation. In line with the essential role of YAP signaling in Kras-driven PC development and progression, knockdown of PR55α impeded the anchorage-independent growth, tumorigenicity, and metastasis of PC cells. Our preliminary data also revealed that the stability of PR55α protein is negatively regulated by the tumor suppressor p53 and its target gene FBXL20, a substrate adaptor for the SCF (SKP1-CUL1-F-box protein) E3 ligase. Consistently, we observed that PR55α is elevated in PC specimens, where its expression correlates with poor patient survival. Based on these findings, we hypothesize that loss of p53 function results in the stabilization of PR55α protein, which, in turn, activates the YAP oncogenic signaling required for Kras-driving PC initiation and progression. We will test the hypothesis by three specific aims: Aim 1 to define the mechanism by which PR55α/PP2A promotes YAP activation and the significance of this cascade in oncogenesis and malignant potential of PC, Aim 2 to elucidate how p53 suppresses the level of PR55α, and Aim 3 to delineate the role of the PR55/YAP axis in pancreatic cancer development and progression in the KPC spontaneous mouse model. The work is innovative, as it will illuminate a critical pathway that promotes PC initiation and progression through PR55/PP2A activated YAP signaling, which is novel to both the PP2A field and the PC field. Successful completion of the proposed studies will yield an in-depth mechanistic understanding of the critical contribution of PR55/PP2A complex to the observed cooperation between Kras activation and the loss of the p53 tumor suppressor during PC development and progression. The acquired knowledge not only will improve the understanding of the role of PP2A in cancer but also will contribute to identifying novel therapeutic targets for improving the treatment of PC.

NIH Research Projects · FY 2024 · 2021-08

Project Summary One hallmark feature of congestive heart failure (CHF) is sodium and fluid retention and in combination with neurohumoral activation leads to poor prognosis and high mortality. Sodium-glucose cotransporter 2 (SGLT2) is localized in the proximal convoluted tubule of the kidney and responsible for 90% of glucose reabsorption. Based on this fact SGLT2 inhibitors promoting glucose excretion are widely used to treat type 2 diabetic patients. To date, clinical studies suggest that SGLT2 inhibitors suppress the risk for hospitalization and mortality for heart failure in type 2 diabetic patients. Further, in non-diabetic patients with CHF, SGLT2 inhibitor prevents worsening heart failure and mortality. Multiple mechanisms have been proposed to be involved in the beneficial effects of SGLT2 inhibitors in CHF. Renal sympathetic nerve activation causes sodium and water retention in CHF. Renal denervation (RDN) has been shown to reduce sodium retention in rats and dogs with CHF. Activation of the splanchnic sympathetic nerve leads to volume redistribution such as to contribute to cardiopulmonary congestion in CHF. Our recent evidence in rats with CHF shows that; 1) levels of SGLT2 expression are dramatically increased in the proximal tubules of the kidney; 2) the activity of SGLT2 for sodium retention is enhanced; 3) RDN decreases the levels of SGLT2 expression and SGLT2 activity; 4) norepinephrine upregulates SGLT2 expression and trafficking in the renal tubular cells; and 5) RDN attenuates renal levels of inflammatory cytokines and renal immune cell activation. Based on these data, we will test the hypothesis that elevated sympathetic activation in CHF enhances sodium reabsorption and fluid retention by modulation of SGLT2 expression, trafficking and function. Further, enhanced SGLT2 expression potentiates a vicious positive feedback between renal inflammation and increases in sympathetic activation (both renal and splanchnic nerves) in CHF. In AIM 1 we will determine if enhanced expression/activation of renal SGLT2 contributes to the sodium retention in rats with CHF. In AIM 2 we will determine if RDN or selective afferent renal denervation abrogates the expression/activation of SGLT2, possibly via renal inflammation in rats with CHF. In Aim 3 we will determine if SGLT2 inhibition reduces efferent/afferent renal and splanchnic sympathetic activation in rats with CHF. These aims will be addressed in rats with CHF using complementary methodologies ranging from cellular to the whole animal level, including physiological measurement of sodium balance, volume status, SGLT2 activity, electrophysiological recording, SGLT2 trafficking using molecular biology techniques. The successful completion of the proposed studies will provide significant new information and insight into the contribution of SGLT2 inhibition on renal nerve mediated regulation in altered sodium balance in CHF and the therapeutic benefits of SGLT2 mediated changes in renal nerve mediated sodium and fluid retention, endemic to CHF.

NIH Research Projects · FY 2025 · 2021-08

Project Summary A comprehensive interdisciplinary approach proposed to support the Single Cell Opioid Responses in the Context of HIV (SCORCH) Program Expansion: CNS Data Generation for Chronic Opioid, Methamphetamine, and/or Cocaine Exposures RFA from the National Institute on Drug Abuse (NIDA). We will perform studies on the interplay of HIV pathogenesis and opioid abuse in the gold-standard SIV/nonhuman primate system, using single-cell RNA sequencing (scRNAseq) to assess individual cellular transcriptomes. This will give us obtain unparalleled insight into the viral and drug-induced effects on diverse cell types in brain in regions critical for HIV infection and opioid abuse. In addition, we will also focus on primary targets of HIV and drivers of neuropathogenesis, microglia and brain macrophages, using scRNAseq as well as single-cell Assay for Transposase-Accessible Chromatin sequencing (scATACseq) to gain insight into the epigenetic regulation of gene expression at the single cell level. These will be combined with state-of-the-art sensitive assays for virus in the brain. We will ensure our data adhere to the FAIR (Findable, Accessible, Interoperable, and Reusable) standards, and will work closely with other members of the SCORCH program to share and disseminate data to yield maximal advancement to the field. Our experiments are carefully designed to address the role of virus, drugs, and their interactions, as well as include the clinically relevant scenarios of those taking antiretrovirals to suppress virus, and those who do not. Our rigor and reproducibility are facilitated by our detailed power analysis with proper sample sizes, as well as our experimental design to match the standards of blocking, replication and randomization. The results from these studies will provide novel insights into the CNS effects of HIV and drug abuse.

NIH Research Projects · FY 2025 · 2021-08

Abstract Angioimmunoblastic T-cell lymphoma (AITL) is the most common subtype of peripheral T-cell lymphoma (PTCL) with distinct pathological and clinical characteristics. It is a difficult-to-diagnose and lacks effective therapies due to poor understanding of the molecular pathogenesis. Using genome-wide gene expression profiling (GEP), we have defined a robust molecular classifier for accurate diagnosis, identified oncogenic pathways, discovered its cell-of-origin, and deciphered the role of the tumor milieu in disease prognosis. Using high-throughput genomic analysis, we also identified frequent mutations in epigenetic regulators (TET2, IDH2 and DNMT3A) and regulators of T-cell activation (RHOA and CD28) in AITL. Co-occurrence of TET2 mutations with IDH2 mutation (hotspot: arginine-172; IDH2R172) is unique in AITL compared to other hematological malignancies where they are mutually exclusive, suggesting their cooperative role in T-cell lymphomagenesis. We have generated unique murine models with conditional loss of Tet2 (Tet2-/-) and double mutant (Tet2-/- /IDH2R172K) in CD4+ T-cells, and murine lymphomas with follicular helper T-cell (TFH) phenotype were observed. In addition, we generated several patient-derived xenografts (PDX) of AITL with IDH2R172 and/or TET2 mutations. We hypothesize that TET2 deficiency in T cells alters the DNA methylation profile leading to an altered genetic program favoring TFH cell differentiation and clonal expansion, facilitating transformation with subsequent hits, including IDH2R172 mutation, which will further alter the epigenome and oncogenic pathways in TFH -cell transformation. We will define the precise role of these genetic lesions in T-cell differentiation and in AITL pathogenesis. We also edited normal human CD4+ T-cells using CRISPR/CAS9 to generate TET2-/- T-cells for functional analysis and cross-validation of murine models. SinceTET2 mutations are in hematopoietic stem/progenitor cells (HSPC) in some AITLs, interactions between TET2 deficient T- and stromal cells during lymphomagenesis will be explored. We will test the efficacy of therapeutic interventions, including demethylating agents for TET2 deficiency and IDH2 inhibitors and/or glutamine depletion for TET2/IDH2 double mutant tumors in murine models and PDX of AITL. To accomplish the objective, three aims are planned: Specific Aim 1: To determine the mechanisms by which Tet2 deficiency mediates T-cell transformation and the role of Tet2 mutation in stromal cells in lymphomagenesis. Specific Aim 2: To delineate the mechanisms of combined Tet2 and IDH2R172K mutations in AITL pathogenesis. Specific Aim 3: To evaluate therapies targeting oncogenic mechanisms mediated by Tet2 and IDH2R172 mutations. Our long-term research goal is to define the pathobiology of AITL, through integrated functional epigenomic approaches using in vitro modified human T-cells, patient samples and relevant murine models, thus identifying promising novel targets for treatment that may be tested pre-clinically in PDX models.

NIH Research Projects · FY 2024 · 2021-08

Abstract Compassionate and effective pain management after surgery represents a foundation of humane medical care. Opioids still present the mainstay of post-surgical pain therapy: more than 80% of patients are prescribed opioids for use following surgery. During one year in the U.S., more than 17% of Americans fill at least one opioid prescription, and, per prescription, the average daily amount exceeded 45 milligram morphine equivalents. In addition to common side effects, serious risks of opioids include addiction, abuse, and overdose. Prescription overdose deaths contribute to more than one-third of all opioid overdose deaths and remain five-fold higher than in 1999. Over-prescription of opioids remains extremely common, with up to 90% of patients after common surgical procedures reporting leftover opioids. Unused opioids are stored in unsecured locations in 75% of cases and can create a reservoir that fuels the ongoing opioid epidemic. Most large-scale efforts, such as the CDC guidelines for prescribing opioids, have focused on chronic opioid use. There is an urgent need to increase access to high-quality, safe, and scalable interventions to reduce our reliance on opioids for effective post- surgical pain management at discharge. A diverse team with expertise in pain medicine, perioperative outcomes, clinical trials, biostatistics, and health economics has been assembled to conduct the Efficiency And Quality In Post-Surgical Pain Therapy After Discharge “EQUIPPED” study. Our central hypothesis is that pain therapy after surgery can and should be prescribed in a safer, patient-centered fashion, as opposed to current “one-size-fits- all” methods. To test this hypothesis, three aims are proposed: Specific Aim 1 will test a provider-facing EHR- based decision support tool that suggests outpatient opioid prescriptions based on inpatient opioid requirements. This aim will utilize a pragmatic randomized cluster multiple crossover controlled trial design in about 39,000 patients in four hospitals. Specific Aim 2 will test a patient-facing health informatics app that encourages non- opioid pain management strategies. This aim employs a randomized controlled trial design in 600 surgical patients. Two separate clinical trials are proposed because of different outcomes (amount of opioids prescribed vs. opioids used) and scopes of implementation (multisite vs. single site). The provider-facing tool will be incorporated into the trial of the patient-facing app using a 2x2 design that facilitates a detailed evaluation of the individual effects of these interventions, as well as any interactions when using the interventions together. Finally, Specific Aim 3 will include a cost-effectiveness analysis and compare the individual and combined cost- effectiveness of a provider-facing and a patient-facing intervention. Completion of this project will demonstrate that the amounts of opioids prescribed and opioids taken after discharge following surgery can be reduced while ensuring effective treatment of pain. Increasing access to safe, affordable, and high-quality pain therapy for surgical patients will drive the positive impact of this application, which directly addresses Special Emphasis Notice: AHRQ Announces Interest in Health Services Research to Address the Opioids Crisis (NOT-HS-18-015).

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Mononuclear phagocytes (MP; monocytes, macrophages, dendritic cells. and microglia) serve as human immunodeficiency virus type one (HIV-1) reservoirs, sites of viral persistence and latency, and inducers of end- organ disease. All are commonly linked to HIV-1 pathobiology. However, the key relevance of the MP viral reservoir rests in the central nervous system (CNS) of those people living with HIV (PLWH). In those PLWH and receiving antiretroviral therapy (ART), the evidence for the size, scope, and disease relevance of the CNS viral reservoir remains under appreciated. The discordance between laboratory MP infection and tissue persistence in an infected human host is also not yet known. MP can have an extended life span and possess self-renewing potential, and as such, are likely more relevant in disease than currently appreciated. Evaluation of the significance of MPs, in general, and the microglia specifically will help define the importance of MPs during natural infection. For the CNS specifically, HIV-1 enters the brain soon after infection and replicates in perivascular macrophages and MGL along with limited numbers of astrocytes. Viral set point and timing of ART initiation determines the latent reservoir size; each affects the efficiency of any eradication strategy. Knowledge of the viral dynamics, CNS viral invasion, susceptibility to MP infection, and composition of CNS HIV reservoir will facilitate effective therapeutic interventions. To each of these ends, we will employ novel techniques to study the MP HIV-1 reservoir in laboratory systems and in a newly developed human microglial mouse model of human disease. We will use basic and applied MP biology, theranostics, novel ART nanoformulations, molecular and cellular biology, and our unique animal model to study the means to eliminate viral infection at the subcellular, cellular, and tissue level with newly designed and novel therapeutic methods that include gene therapy strategies. Our aims are to determine the efficiency of viral suppression by native and nanoformulated ART (at subcellular level), assess the breadth of the CNS viral reservoirs against viral set points (defined by the initiation of ART), and to explore combination strategies for HIV-1 elimination in a new developed humanized microglial mouse.

NIH Research Projects · FY 2025 · 2021-08

Scientific Abstract Hemoglobin, the oxygen carrying protein expressed in erythrocytes, can be glycated following elevations in blood glucose. Some amino acids, such as the N-terminal valine of the hemoglobin beta chain are highly susceptible to glycation in diabetic patients. This specific glycated isoform, termed HbA1c, has been used by clinicians as an overall picture of a diabetic patient’s ability to control their glucose over a 3-month period and as an indicator for future cardiovascular risks. Recently, it was observed that the alpha chain of hemoglobin, but not the beta chain, is expressed in endothelial cells lining arteries where it interacts with endothelial nitric oxide synthase (eNOS) to modulate nitric oxide (NO) release. Hemoglobin alpha is known to be glycated at a number of sites, including one in the putative eNOS interaction domain. Since it is well recognized that vascular dysfunction underlies many of the pathologies in diabetic patients, it was hypothesized that the hemoglobin alpha expressed in the endothelium will have aberrant function in diabetes mellitus, likely due to a glycation event. The aim of the current proposal is to examine the role of hemoglobin alpha and any possible glycated forms of hemoglobin alpha in the endothelium of a murine model of diabetes. Using pharmacological and genetic approaches, the interaction between hemoglobin alpha and eNOS will be disrupted and the influence on the development of vascular dysfunction will be explored. This work has the potential to identify both a novel biomarker of vascular risk and also a potential therapeutic target for pharmacological treatments.

NIH Research Projects · FY 2025 · 2021-07

Project Summary The overall objective of this project is to determine the impact of social stressors on epigenetic age acceleration and chronic health disparities and to test whether the social environment, individual health behaviors, and race/geography/SES modify or mediate the association between traumatic stress and health (disparities), directly or indirectly through biologic age acceleration. We expect that the trauma burden will impact chronic diseases through DNA methylation (DNAm) age acceleration. The WaTCH cohort is an important, highly trauma- exposed sample of women uniquely poised for a third wave of data collection that continues assessing trauma exposure, depression, PTSD, social context, physical health, and collection of an additional blood specimen eight years after the baseline to examine of DNAm age. Aim 1 will investigate disparities in psychological health (PTSD, depression), physical health (diabetes, hypertension, and cancer), and DNAm age acceleration as women age, particularly as influenced by cumulative trauma burden. Aim 2 will examine the influences of contextual variables, including social capital and financial strain, that may mediate or moderate the effects of cumulative trauma burden on adverse psychological and physical health outcomes. Aim 3 will explore the mediating effects of DNAm age acceleration on physical and mental health outcomes. The proposed study will use the WaTCH cohort of 2800 women exposed to the Deepwater Horizon Oil Spill in coastal Louisiana in 2010. It will entail the third wave of data and biospecimen collection and incorporate data already gathered through two previous waves of data collection. Self-reported health data on demographic, income and financial stressors, oil spill exposure, neighborhood context, social capital, health behaviors, trauma history, psychological symptoms, and physical health will be collected through telephone interviews. Repeat blood samples will be collected from up to 1058 women with baseline samples.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Since the start of the HIV/AIDS epidemic, it has been recognized that drug abuse is strongly linked to contracting and transmitting HIV, as well as more severe consequences. Factors influencing the susceptibility and disease progression are still unclear. Viral reservoir establishment occurs early in acute HIV infection and reaches a set point within the first two months of infection, which in turn determines the size of the reservoir in the chronic stage. HIV enters the brain right after infection and replicates in macrophages, microglia, and to a small extent in astrocytes. Increased susceptibility in substance use could affect the size of the central nervous system (CNS) reservoir, which may influence spontaneous virological remission and viral rebound kinetics. CNS viral reservoir size may also determine the HIV- associated neurocognitive disorder (HAND) severity, viral recurrence, and rebound. Until now, there was no means to study the viral dynamics, CNS viral invasion, susceptibility to MP infection in the brain, and the reservoirs of HIV infection so that interventions can be developed effectively. A small animal model, like the humanized mouse, provides great advantages due to manipulations using host/human cells, as well as the possibility of establishing chronic infection by HIV itself. The humanized microglial (hMGL) mouse model is reconstituted with the human immune system and human glial cells, which allows natural progression of CNS infection and disease in relation to peripheral HIV infection. Herein, we propose to deploy our hMGL mouse to elucidate the mechanisms of HIV-1 invasion, persistence, and neuropathogenesis for substance use. Our objective is to define the CNS viral reservoir and investigate changes in the brain viral reservoir as a consequence of substances of abuse with an overarching goal of viral eradication. Our aims are, 1. to investigate the effect of cocaine on the CNS viral invasion, reservoir establishment, and viral latency during chronic infection in humanized microglial mice, 2. to determine the host genetic signatures that accelerate the CNS viral reservoir and assess the influence of antiretroviral therapy (ART) on glial homeostasis, and 3. to study HIV and SUD-induced neuropathology using a multimodal approach. The complete characterization of this humanized mouse model in establishing linkages to human disease, and demonstrating a comprehensive evaluation of the CNS reservoir during cART, HIV infection, and drug abuse will prove to be a significant value to the neuroHIV field.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal and is projected to become the second-leading cause of cancer related deaths in the US by 2030. Conventional (genotoxic) chemotherapy approaches that make up the current standard of care are mostly ineffective and prolong survival of advanced PDAC patients by less than one year on average. Similarly, small molecule drugs targeting aberrantly activated oncogenic signaling pathways have shown disappointing clinical results and accordingly have failed to gain FDA approval for PDAC. An alternative strategy to these two approaches is to exploit metabolic dependencies that are unique to malignant cells by virtue of their deranged cellular metabolism. While there are well-characterized resistance mechanisms to genotoxic and targeted therapies, deprivation of certain nutrients critical for proliferation of malignant cells appears to be an insurmountable barrier for cancer progression. However, cells have redundant means of acquiring these critical nutrients, and so inhibition of a single metabolic enzyme is generally not sufficient to deny them to cancer cells. Thus, combinatorial blockade of multiple metabolic pathways could be required to impose deficiency of key metabolites on malignant cells. Pyrimidine nucleotides represent a class of metabolites that has been shown in numerous studies to be essential for PDAC and a host of other malignancies. Importantly, there are several clinical grade inhibitors of pyrimidine synthesis enzymes that have shown preclinical promise as anticancer agents. However, these drugs have uniformly failed to show efficacy in clinical trials in which they were used as monotherapy against various malignancies. One potential explanation for this is that there are two major pathways by which cells generate nucleotides, termed the de novo and salvage pathways, and these inhibitors block the key de novo pathway enzyme dihydroorotate dehydrogenase (DHODH), thus leaving the salvage pathway fully intact. We’ve characterized the response of various PDAC cell lines to the DHODH inhibitor brequinar (BQ). We found that some PDAC cell lines are highly resistant to BQ in cell viability assays compared to their more sensitive counterparts. We then screened some 350 known kinase inhibitor compounds to probe for any that could restore BQ sensitivity in our resistant PDAC cell lines, and this nominated the preclinical BTK inhibitor CNX-774 as the strongest hit. Follow-up studies have shown that combined BQ/CNX-774 treatment leads to profound cell viability loss and pyrimidine depletion, compared to either drug alone, in BQ-resistant PDAC cells. Furthermore, we have strong evidence that CNX-774 is acting in an off-target manner to inhibit pyrimidine salvage. Thus, the goal of this study is to uncover the mechanism by which CNX-774 is sensitizing PDAC cells to BQ and determine if this drug combination is efficacious in our preclinical PDAC mouse models. Our goal is to provide preclinical support for this metabolic combination therapy as a potential PDAC treatment.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY: Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by the cardinal motor symptoms of resting tremor, hypokinesia, muscle rigidity and bradykinesia. To date, dopamine replacement therapy using the dopamine precursor levodopa, or L-DOPA, remains the gold standard treatment for the motor symptoms in PD. However, development of L-DOPA-induced dyskinesias (LIDs) represents a major dose-limiting adverse effect associated with the long-term treatment of PD using L-DOPA. For example, approximately 10% of PD patients per year develop LIDs within the first 7-8 years of L-DOPA treatment. Moreover, there are no effective treatments for either preventing the development of LIDs or reversing already established LIDs in PD patients. To date, there remains a critical unmet need to develop novel therapeutic approaches for the complications associated with chronic L-DOPA treatment in PD. We have recently discovered a series of dopamine 4 receptor antagonists which are potent and selective for the D4 receptor over the other dopamine isoforms, and a wide selectivity panel. In addition, we have shown that a prototypical compound from this scaffold was capable of producing antidyskinetic action in a mouse model. Subsequently, we have discovered two additional novel scaffolds that show excellent potency and selectivity as D4 antagonists. In this proposal, we will improve and optimize our lead scaffolds in order to improve the ADME properties (metabolic stability) while maintaining the potency and selectivity. In order to develop these best-in-class compounds, we will utilize an iterative medicinal chemistry approach and integrated DMPK studies which will allow us to evaluate potency and selectivity, but also the in vitro and in vivo DMPK properties of newly made compound in a timely manner. The advanced D4 receptor antagonist compounds will then be evaluated in an in vivo animal model of LIDs. These selective D4 receptor antagonist will offer a unique opportunity to help advance the field toward a first-in-class therapeutic agent.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Inhalation of aerosolized dusts from urban, rural, and farming environments can trigger harmful airway inflammation and injury; over time, continual exposure to these particulates increases one’s risk for developing inflammatory airway diseases. While dust exposures negatively impact lung health, factors contributing to protection versus susceptibility to lung disease following these continual inhalational exposures are unclear. A recently discovered class of specialized pro-resolving lipid mediators (SPM) derived from omega-3 fatty acids regulate lung inflammation, immunity, and repair, and are likely key to the beneficial effects of diets high in omega-3 fatty acids. Our previous investigations identified that the omega-3 fatty acid docosahexaenoic acid (DHA) and its lipid metabolite maresin-1 (MaR1) mitigate airway inflammation from acute and repetitive organic dust exposure, mediated in part by macrophage activation and pro-repair activities on the airway epithelium. Our exciting new data identify that omega-3 fatty acids and MaR1 can activate IL-22 signaling in lung macrophages. IL-22 signaling promotes mucosal immunity and epithelial barrier integrity, and its activation in the presence of these bioactive lipids may be key to their protective effects. Furthermore, our novel finding of IL-22 signaling in macrophages challenges current dogma regarding the activation and regulation of this pathway. The goal of this proposal is to investigate the role of omega-3 fatty acids in promoting pro-repair IL-22 signaling in the lung following dust exposures. We hypothesize that omega-3 fatty acids and SPM promote lung recovery following particulate matter exposures by inducing alveolar macrophage IL-22 production that subsequently promotes alveolar macrophage pro-resolution polarization and lung epithelial repair. To test this hypothesis, in Aim 1, we will establish the impact of omega-3 fatty acids and IL-22 on lung recovery following dust exposure. In Aim 2, we will evaluate the role of omega-3 fatty acids and IL-22 in epithelial repair and mucosal immunity during dust exposure. In Aim 3, we will identify how SPM and IL-22 signaling impacts lung macrophage polarization. Together, our studies will identify how omega-3 fatty acids modulate susceptibility versus resilience to dust exposures, including a novel protective mechanism via activation of macrophage IL22 signaling to promote tissue repair and mucosal immunity. We expect our studies’ findings to guide novel treatment strategies for lung disease.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT: Fatty liver (steatosis), characterized by an accumulation of lipids in hepatocytes, is one of the earliest pathological changes in the progression of alcohol-associated fatty liver disease (AFLD). Pathophysiological mechanisms involved during development of AFLD are complex and multifactorial, including gut, pancreas and adipose tissue dysfunctions that reportedly affect liver pathology. Accumulating evidence has demonstrated that the crosstalk between these organs are regulated by peptide hormones. Especially relevant to this proposal is the growing interest in understanding the role of gut hormones in organ interactions and in the development of AFLD. Among all the gastrointestinal hormones, the stomach-derived ghrelin is the one of the hormones that significantly increases with chronic alcohol exposure in humans and experimental animal models. In our recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in the circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Concomitantly, chronic alcohol treatment to rats increases serum levels of the gut hormone, glucagon-like peptide-1 (GLP-1) while decreasing liver-expressed antimicrobial peptide-2 (LEAP-2) and adiponectin levels. Interestingly, these pathological changes were not altered in ethanol-fed ghrelin receptor knockout (GHS-R KO) rats, which were also resistant to steatosis development. Collectively, these results indicate a fundamental role of an alcohol-induced ghrelin increase in affecting multiple organs, such as the gut and adipose to modulate GLP-1, insulin, adiponectin and LEAP-2 activity/levels, to ultimately lead to the development of AFLD. To study these effects, we present the following hypothesis: Alcohol-induced increase in serum ghrelin levels directly (i) inhibits GLP-1 hormone-mediated energy metabolism in hepatocytes and (ii) modulates adipose metabolism to increase adipose lipolysis and decrease adiponectin secretion, both of which contribute to hepatic steatosis. Furthermore, ghrelin also enhances its effects by lowering the levels of LEAP-2, recently discovered endogenous ghrelin antagonist peptide that reduces the ghrelin receptor (GHS-R) mediated signal transduction. We will utilize a variety of state-of-the art technologies and innovative biological concepts to explore our hypothesis in three specific aims: 1) Characterize the role of ghrelin in modulating the gut-derived GLP-1 hormone-mediated gut-liver crosstalk during the development of AFLD; 2) Characterize the specific contribution of ghrelin in modulating the adipose-liver axis in the development of ALFD; and 3) Examine the effect of alcohol- induced ghrelin increase on LEAP-2 expression and characterize its role in modulating hepatic steatosis. Successful completion of the proposed studies will aid mechanistic insights into understanding ghrelin’s role in modulating the gut, adipose and liver axis to promote the development of alcoholic steatosis.

NIH Research Projects · FY 2025 · 2021-04

The University of Nebraska Medical Center will continue to leverage the trust and cooperative spirit we have garnered working with tribal schools and communities in Nebraska and South Dakota. We will develop, implement, and evaluate science curriculum, outreach activities, and training experiences targeting Native American students and their teachers in grades K-12. The long-term goals of this project are to promote student interest in the sciences, foster a more science-literate public, and ultimately to increase the number of Native Americans entering health and science careers. Improving science and math instruction in the classrooms serving our Native American partners is key to this project. This objective will be accomplished by creating and adapting hands-on, age-appropriate lessons. Through summer workshops, mentoring, and in-service education, teacher support will facilitate the implementation of novel science and math education strategies for use in tribal schools. Student engagement will be enhanced through summer experiences from science camps for middle school to longer-term enrichment programs for select high school students. Reaching beyond the classroom to parents and communities is also critical to the success of this project. Community programs will be designed to give parents and elders the sense of excitement that students feel when doing science. Ancillary benefits will include promoting educational opportunities, healthy living, and improving health literacy. Professional evaluations will be made at all stages, with major emphasis being placed on evaluating the educational and community impact of the project. Research techniques will include baseline and post measures of attitudes and subject content, participant evaluation questionnaires, and student retrospective pretests. Advancing the health of Native American communities is the ultimate aim of every aspect of this project. Improved science teaching and heightened awareness of health careers will encourage students to enter these careers and hope to bring these skills back to their communities. Public outreach that increases health literacy and healthy living will promote better personal health decisions.

NIH Research Projects · FY 2025 · 2021-04

Muco-obstructive airway diseases including asthma, COPD, cystic fibrosis, and non-CF bronchiectasis have diverse genetic and environmental origins, but have certain common features that includes pathologic epithelial changes referred to as mucous metaplasia. Airway secretory cells differentiate into mucous cells with a goblet cell morphology packed with mucin granules containing MUC5AC and, to lesser extent, MUC5B. These airway diseases are characterized by frequent exacerbations due to mucous hypersecretion and blockage of the airways that leads to loss of lung function, hospitalization, and risk of death. While many of the factors that cause mucous metaplasia have been identified, little is known about how it resolves. Autophagy is a key cellular protein recycling system that degrades proteins in response to nutrient deprivation, inflammation, and infection. We have spent the last several years studying the role of autophagy in airway disease using models with genetic deletions of a key autophagy regulatory genes. In this application, we propose a new paradigm in which mucin granule degradation contributes to resolution of mucous metaplasia through the action of autophagy. Three key findings in our preliminary data support this hypothesis: First, autophagy deficient mouse and cell culture models accumulate more cytoplasmic mucin granules during mucous metaplasia and particularly during resolution. Second, mucous metaplasia is associated with mTOR activation and increased epithelial metabolism which is then down-regulate during resolution. We propose that this shift in metabolism is the key trigger initiating mucin degradation during resolution. Third, mimicking this shift in metabolism with mTOR inhibitors leads to autophagy activation and mucin degradation in human airway epithelial secretory cells. To test our hypothesis that autophagy leads to degradation of mucin granules, we propose three research aims: First, we will determine how mTOR signaling contributes to metabolism change in the secretory cell and ultimately to autophagy-mediated mucin degradation. Second, we will characterize the importance of autolysosome-lysosome fusion during mucous metaplasia resolution by examinig vesicle trafficking, lysosome biogenesis, and lysosome proteolytic function. Third, we will explore mucin degradation as a therapeutic strategy in models of muco-obstructive airway diseases. These findings can provide the framework for a new therapeutic strategy to hasten the resolution of airway disease exacerbations.

NIH Research Projects · FY 2025 · 2021-04

Abstract This project contains a highly collaborative investigative team with interdisciplinary expertise with significant potential impact for HIV/AIDS prevention. The proposal includes pharmacologic, virologic, animal and product development studies designed to halt disease transmission through novel long-acting (LA) antiretrovirals. These are named LA slow effective release antiretroviral therapies (LASER ART) designed to facilitate HIV-1 prevention by intense bench to the clinic translational studies. Innovative interdisciplinary approaches contain a detailed research plan, extensive preliminary and broadly published supportive data. Creativity and innovativeness are offered placed at higher risk than a conventional research project. The work builds on the development of parenteral nanoformulations of chemically modified antiretroviral drugs designed to improve adherence. The drugs are currently offered once/day in pill form but will be converted to up to once a year administration. Support from expert pharmacologists and pharmaceutical scientists with University researchers are operative. The drugs include dolutegravir (DTG), emtricitabine (FTC) and tenofovir (TFV) created to extend their apparent drug half-life, efficacy and abilities to target viral reservoirs. They are, in measure, DTG and FTC and TFV prodrug + nucleotide (ProTides) designated “N” for nanoformulation, “M” for esterification and “P” for ProTides. The created NPFTC and NPTFV and NM2DTG demonstrate sustained plasma and tissue drug concentrations of > 90% inhibitory concentration from months to a year. Based on encouraging results, we seek funds to facilitate large scale development that would facilitate future human studies. The final formulations would be characterized by sustained prodrug hydrolysis with reduced injection volumes. The pathway forward follows established partnership with the Clinton Health Access Initiative and oversight by ViiV Healthcare and Gilead Sciences. The overarching goal is safety, reproducibility and “scale-up” that follows US Food and Drug Administration-approved current good manufacturing practices (cGMP). The specific aims are each supported by extensive published data sets forged through the multidisciplinary research. Creation and characterization focus on prodrug formulations, toxicology, and pharmacokinetics profiles follow a safe developmental action plan. The work is facilitated by a fully operational cGMP facility and rhesus macaque validations. The lead formulation will be developed with our CHAI partners. We posit that the creation of LASER ART DTG or FTC and TFV will have a profound impact on HIV prevention.

NIH Research Projects · FY 2025 · 2021-04

Objective: More effective treatment options are needed across all clinical stages of colorectal cancer (CRC), from eliminating residual disease after surgery, improving surgical candidacy and developing more effective treatment options for metastatic disease. Targeted radionuclide therapy (TRT), which targets tumor-specific biomarkers, has been one appealing approach in the pursuit of effective cancer therapeutics. The development of low-molecular-weight TRT agents is particularly attractive due to their rapid targeting and non-target clearance properties. However, for many investigated low-molecular-weight TRT agents, the short residence time in tumors due to inherently high clearance rates of the agents and their metabolites inhibits clinical translation. The purpose of this proposal is to design a CRC therapeutic agent capable of targeting the neurotensin receptor subtype 1 (NTSR1), a receptor found to be overexpressed in large segments of the CRC patient population. Specifically, by incorporating irreversible cysteine protease trapping agents (CPTAs) into the structure of NTSR1-targeted agents (NTSR1TAs), we seek to design TRTs that are capable of forming high molecular weight intracellular adducts. Through this retention mechanism, the 177Lu-labeled, CPTA-incorporated, NTSR1TAs (177Lu-CPTA- NTSR1TAs) will exhibit substantial increases in radiation dose delivery leading to enhanced therapeutic efficacy. Also, as part of this grant, we seek to gain a clearer understanding of the in vivo adduct binding partners, adduct half-lives and tissue/cellular localization associated with this trapping mechanism. Specific Aims: (1) Synthesis and Initial Biological Performance of 177Lu-CPTA-NTSR1-targeted Agents; (2) Examine In Vivo Adduct Protease Profiles, Adduct Half-lives and Tissue Distribution; and (3) Therapeutic Evaluation of Optimized 177Lu-CPTA-NTSR1-targeted Agents Study Design: The first aim of this proposal is to examine pathways to further improve the design of CRC residualizing 177Lu-CPTA-NTSR1TAs by 1) refining the construct to reduce T/K radiation dose ratios; 2) exploring the impact of CPTAs with varying selectivities; and 3) examining different cysteine protease inhibitor classes. After synthesis, initial assessments regarding the in vitro and in vivo performance of the177Lu-CPTA-NTSR1TAs will be performed. In the second aim, a more detailed biological evaluation will be carried out in which adduct- binding partners are identified and quantified, tissue protease expression levels ascertained, adduct half-lives are estimated and cellular distribution (percent intracellular, membrane or extracellular) determined. Lastly, lead TRT candidates will be examined in advanced CRC mouse models to determine the best candidates to move forward to the maximum tolerated dose and therapeutic efficacy studies. At the end of this project, we anticipate being able to identify a candidate to advance towards FDA investigational new drug approval.