University Of Florida

NIH Research Projects · FY 2025 · 2022-08

Project Summary: Despite standard of care with maximal surgical resection, external beam radiotherapy, and chemotherapy, patients with glioblastoma (GBM) live little more than 18 months after diagnosis; these outcomes necessitate development of new targeted therapies. To address this gap, our group developed a novel vaccine formulation that can unlock anti-tumor immunity within hours. By layering tumor mRNA into a multi-lamellar nano-lipid formulation (for systemic administration), we can make tumor antigens appear like a systemic viral infection. Our multi-lamellar design delivers increased antigenic load (per particle) triggering potent innate activation which then facilitates adaptive effector responses. RNA-lipid nanoparticles (RNA-LPs) activate systemic/intratumoral dendritic cells (DCs), upregulate critical innate gene signatures in the glioma microenvironment, and induce glioma-specific T cell immunity. In murine tumor models resistant to immune checkpoint inhibitors, RNA-LPs induce robust anti-tumor efficacy with long-term survivor benefits. We have previously demonstrated safety of RNA-LPs in acute/chronic murine GLP toxicity studies and launched a large animal canine glioma trial (IACUC#201609430). Our canine trial demonstrated that RNA-LP administration is feasible, safe and immunologically active with improvement in overall survival in pet dogs with terminal gliomas (compared with historical controls). We have since received FDA-IND approval (BB-IND#19304, Sayour) for first- in-human studies (NCT04573140) in patients with GBM. The purpose of this study is to assess the safety, maximum tolerated dose (MTD), and immunogenicity of RNA-LPs vaccines in newly-diagnosed adult GBM patients. We hypothesize that RNA-LPs will mediate systemic immune reprogramming of GBM unlocking immunotherapeutic activity. Our SPECIFIC AIMS are: 1. Conduct phase I study evaluating safety, MTD and immunogenicity of RNA-LPs against GBM. a. Characterize systemic immune response in patients prior to receiving RNA-LPs and effect change following vaccination. i. Identify immunologic phenotype of myeloid and lymphocyte subsets (naïve, effector, memory, regulatory) and NK cells in patients with GBM at diagnosis and throughout therapy. ii. Elucidate changes in cytokine profile and pathogen-recognition receptor (PRR) activation status in patients with GBM during/after RNA-LP vaccines. iii. Identify potential tumor specific antigens as vaccine candidates through whole exome sequencing, RNA-seq, and neoantigen prediction analysis. b. Establish memory recall T cell immunity in vaccinated patients c. Determine if magnitude and persistence of anti-tumor innate and adaptive immunity correlates with clinical outcome.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Severe infection and sepsis accelerate cognitive decline in older Americans, especially those with incipient Alzheimer's disease (AD). Sepsis is known to induce a systemic inflammatory `cytokine storm', which wanes with time. However, this response often persists even after infection resolution in patients. We hypothesize that this systemic cytokine storm can potentially affect brain neuropathology in both pre-symptomatic AD patients and in older adults with no known cognitive deficits. In preclinical AD models, both amyloid and tau protein accretion and pathology are influenced by systemic inflammation. Less is known about the cognitive decline in sepsis survivors without incipient AD, known as sepsis-associated encephalopathy (SAE), and whether this is related to development of amyloid and tau neuropathology. Our overarching hypothesis is that sepsis and CCI induce unique local and systemic immunological responses that directly influence murine AD- and SAE-related pathology. We hypothesize that in incipient or prodromal AD, sepsis would specifically exacerbate brain health by exacerbating amyloid and tau neuropathology, while in cognitively normal older individuals, SAE outcomes would be exacerbated by aging pathways. We will test these hypotheses using 4 specific aims. In Aim 1) we will evaluate whether age plays a critical role in driving sepsis-induced neurodegeneration and loss of cognition (SAE) in wild-type mice. Here we propose to employ a survivable cecal ligation and puncture model of polymicrobial sepsis with daily chronic stress (CLP+DCS) in 6 month (mo) young and 18 mo older adult C57BL/6 (B6) mixed sex mice. Mice will be euthanized at 4 or 8 weeks post-sepsis for inflammatory and neuropathologic evaluation, which will include analysis of neuronal death, and brain inflammatory changes. Simultaneous spatial transcriptomic and proteomic profiling (NanoString GeoMx™) will allow assessment of brain region-specific alterations in response to the peripheral cytokine storm. Plasma will be analyzed for inflammatory cytokines and selected alarmins. Mice will also undergo cognitive assessment following their septic insult. In Aim 2) we will evaluate whether CLP+DCS-induced systemic inflammation alters Aβ deposition. Here we will use two APP transgenic mouse models – the fast progressing TgCRND8 mice and the slow progressing Tg.PrP HuAβ (APPsi) mice - to assess how systemic cytokine storm modulates amyloid deposition. In Aim 3) we will evaluate whether systemic inflammation exacerbates tau pathology. We will assess whether sepsis induces tau pathology in AD- relevant brain areas in the PS19 mice. Finally, in Aim 4) we will test whether prophylactic manipulation of the immune system alters SAE or AD-associated pathologies in selected animal models. Using an AAV-directed decoy sIL-10R, we will test whether suppressing IL-10 systemically or in the brain attenuates neuropathologic/behavioral outcomes. Additional mice will receive metformin orally during sepsis as it may be neuroprotective. This study will provide a mechanistic under-standing of sepsis-induced pathology, as well as differences in older adults with and without pre-existing AD.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Materials for applications in healthcare and medicine usually come from two main groups (a) synthetic polymers specifically designed to achieve a certain goal or (b) naturally derived biopolymers that are leveraged in their native or slightly modified state for a specific goal. The advantage of being a synthetic chemist is that technically, if you can synthesize it, the possibilities are infinite, but the downfall is that often solvents or portions of the polymer cause cytocompability issues or concerns when it comes to translation and implantation in a human. Alternatively, unmodified natural biopolymers, or proteins, have an easier path toward Food and Drug Administration's approval, but lack the customizability afforded in synthesis or chemical modification. Genetic engineering via production of small peptides in bacteria has improved the availability of customizable short peptides, but proteins on the order of hundreds of kilodaltons cannot be produced this way. This is the case for the silk fibroin biopolymers isolated from caterpillars in the Lepidoptera order, where the heavy chain of silk fibroin is known to be over 300 kilodaltons in length. Genetic engineering, using tools such as CRISPR or PiggyBac, provides an avenue for theoretically modifying the sequence of silk proteins, which has been attempted with limited success in the domesticated silkworm, Bombyx mori. However, silk is collected from the cocoon of the B. mori pupae, meaning that the life cycle of this silkworm is interrupted, making it difficult to maintain these modified populations or assess phenotypes in a high-throughput manner. To address this, silk fibroin will be isolated from an entirely different silk-producing species: Plodia interpunctella, or the Indianmeal moth. Under specific conditions, this agricultural pest produces sheets of silk prior to entering the cocooning phase. These easily collectable sheets of silk fibers can then be cleaned, degummed, and regenerated to an aqueous biopolymer solution. Moreover, unlike B. mori, P. interpunctella silk collection does not interrupt the life cycle of the silkworm/moth and these silkworms are easier to stably genetically modify though embryo injections compared to B. mori. In this Maximizing Investigators' Research Award, genetic engineering will be leveraged to modify the silk fibroin protein sequence at the organismal level, adding in new peptide sequences such as mammalian cell binding motifs or sites for human growth factor sequestration. Scale-up of the process will be achieved via transcriptional regulation of silk fibroin as a function of external stimuli such as humidity or pathogens. Together, these two strategies for enhancing the bio-functionality of the silk fibroin protein and the scale-up required for advanced manufacturing of medical devices or materials will be explored. The outcomes of this work include full biophysical, biochemical, and in vivo characterization of these materials through analysis of systemic and local immune responses in vivo, complete characterization of the new biopolymer structures, and investigation of mechanotransduction in these materials in vitro. Future work aims to leverage this new class of biopolymers for specific applications in pharmaceutical delivery, tissue engineering, and muscle rehabilitation.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Acute neural injury from subarachnoid hemorrhage (SAH)-associated cerebral infarction occurs in 30-40% of patients who survive the initial hemorrhage, leads to death and disability, and most strongly correlates with 3- month outcome. There is also a significant rate of long-term cognitive deficits. Current understanding of the pathophysiology of post-SAH cerebral infarction points to injury cascades involving decreased nitric oxide (NO) bioavailability and oxidative stress. Under normal conditions, NO signaling pathways regulate cerebral blood flow by mediating cerebral vasodilation and inhibiting platelet adhesion. However, with SAH, red blood cells lyse and release hemoglobin, which is a spasmogenic. Hemoglobin scavenges NO; stimulates production of a nitric oxide synthase (NOS) inhibitor (ADMA); and generates reactive oxygen species (ROS) and nitrogen species (RNS). We believe the recently-identified peptide hormone adropin is a promising therapeutic target for post-SAH cerebral infarction. Our group and others have shown adropin is abundantly expressed in the brain, regulates the endothelial nitric oxide synthase (eNOS) pathway, correlates with markers of oxidative stress, and reduces brain endothelial permeability in response to simulated ischemia. Our hypothesis is that adropin confers protection against acute neural injury from post-SAH cerebral infarction. Our overall goal in this proposal is to demonstrate the protective role of adropin in SAH and investigate the underlying molecular and cellular mechanisms of this protection. Our preliminary data support this hypothesis by showing that SAH decreases brain adropin expression, and that endogenous adropin overexpression by transgenic mice with a β-actin-driven adropin transgene (AdrTg) or treatment with synthetic adropin in the SAH model reduces cerebral edema, preserves tight junction protein expression, abolishes microthrombosis, increases eNOS phosphorylation, prevents cerebral vasospasm, and inhibits neuronal apoptosis. Aim 1 is to determine whether adropin confers protection against acute neural injury after SAH when given in a clinically translatable timepoint after SAH. In Aim 2, we will study mediators that regulate Enho gene expression in the setting of SAH. In Aim 3, we will study whether adropin neurovascular protection is mediated via eNOS activity. We expect this study will provide novel knowledge on adropin-mediated protection against acute neural injury after SAH. The significance of this study is that it is directly translatable and the first to investigate the fundamentals of adropin regulation in brain endothelial cells after SAH. This study will provide the preclinical data for a Phase 1/2 human clinical trial in SAH patients.

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract DNA is frequently damaged by exogenous sources ranging from exposure to UV light to toxic chemicals in the environment. To fix the damage caused by these agents and maintain genomic stability, cells have multiple efficient DNA repair mechanisms. Some damage, though, will inevitably escape repair if the burden of damage is too high. Unrepaired DNA damage can block DNA synthesis and have serious consequences for the cell and for human health. A study by Brown et al. used azidothymidine (AZT) as a tool to block replication in E. coli to discover essential genes for resolving stalled replication forks. AZT is a thymidine analog that can be incorporated during synthesis and prevents primer extension, causing replication to stall and single-strand DNA gaps to form. Two genes, yoaA and holC, were discovered to be vital for resolving stalled DNA replication in AZT treated E. coli cells. The yoaA gene encodes for an XPD/Rad3-like helicase. The four human XPD/Rad- 3 like helicases (FANCJ, XPD, RTEL1, and CHLR1) contribute to genomic stability and if compromised, can cause various genetic diseases and an increased risk of cancer. The holC gene encodes for chi, which is a part of two different complexes. Chi is an accessory subunit of the DNA polymerase III clamp loader and forms a complex with the holoenzyme. Chi also binds YoaA to create a functional YoaA-chi helicase. Chi is known to bind single-stranded DNA binding protein (SSB) and this interaction is necessary for resolving lesions that stall replication. SSB is an essential protein found in all domains of life, coats single-stranded (ss) DNA, and interacts with over a dozen DNA repair and replication proteins. How YoaA, chi, and SSB work together to resolve damage that halts replication is unknown. Therefore, this fellowship aims to characterize SSB interactions with YoaA-chi with biochemical techniques to understand this novel repair pathway. It is hypothesized SSB regulates the ability of YoaA-chi to unwind double-stranded DNA to resolve lesions at the replication fork based on preliminary data which shows that the helicase activity of YoaA-chi is decreased in the presence of SSB. How SSB binds YoaA-chi will be elucidated, be it either by the known location on chi or by a new interaction possibly on YoaA (aim 1). Because SSB regulates a variety of DNA-binding proteins through various mechanisms, several facets of YoaA-chi that SSB could regulate will be investigated. It will be determined if SSB changes the substrate affinity of YoaA-chi (aim 2) or the helicase activity of YoaA-chi (aim 3). This will be the first study into how SSB regulates YoaA-chi and the contribution these proteins have in a novel DNA repair mechanism. This research will also provide significant contributions in my training to become an independent biochemist and the environment at the University of Florida will allow me to be successful.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Studying cancer across a diverse array of species provides a unique opportunity to interrogate factors underpinning cancer initiation and progression and facilitating the modeling of new therapeutic targets in the setting of spontaneous tumors complicated by comorbidities and metastases. While there is extensive reporting of the frequency and diversity of tumors in animals from zoos, it has not been systematically linked to the plethora of genomic resources available. To facilitate the transition of comparative oncology studies from human plus one or two other species, to pan-mammalian analyses, we propose building a pan-mammalian tumor compendium and portal to easily disseminate our resource. We will develop and apply machine learning tools to detect patterns of cancer emergence and cancer resistance in human and non-human mammalian tumors. Our approach provides opportunities to leverage the largely understudied mammalian tumor data jointly with human data to identify reciprocal links between evolution and cancer resistance. This will allow us to make new human cancer discoveries through integrative analysis of high-throughput biological data in the context of mammalian species. We offer a powerful approach combining high-quality reference genomes and genomic data from hundreds of mammalian species with machine learning, that has the promise to unearth the evolutionary genetic underpinnings that are cornerstones of cancer initiation and progression. We will develop and apply models to identify mammalian tumors that effectively mimic rare human cancers, work with collaborators to acquire tumor samples from strongest identified mammalian models, then sequence and analyze these samples to validate their effectiveness as models of human cancers.

NIH Research Projects · FY 2025 · 2022-08

Abstract: Advancement in high-resolution microscopy has opened unprecedented opportunities to investigate cells and tissues spatially at sub-micron level, via molecular imaging of gene transcripts, proteins or metabolomes. Parallel advances in computer-based hardware technologies and AI/ machine learning (ML) also offer a vehicle to study such multi-omics data in high dimensionality. An outstanding challenge involves a fusion of such data and thorough understanding of the fused data in all possible domains, including in basic science, clinical or pre-clinical studies using model systems, clinical diagnosis, prognostication, and drug discovery. Human Bio-Molecular Atlas Project (HuBMAP) consortium is an avenue for generating high-resolution multi-omics data at single cell resolution using a multitude of spatial molecular omics technologies. Common imaging modality that connects all these data types is brightfield histology microscopy, which is inexpensive and integrates the above-mentioned multi-omics data with clinical decision making. This HIVE Tools proposal aims to develop and implement novel machine learning pipelines to predict cell types and/or states from brightfield histology images using spatial protein- and/or RNA-based technology data with concurrent brightfield histology. This will enable using these spatial omics data as a bridge to link histology with high content single cell data sets and thus create a single exploration space from histology to biomolecules in distinct cell types. As a first step, we will employ select data collected under HuBMAP or generated via this HIVE team using CODEX as well as spatial transcriptomics (ST), and develop the proposed computational pipeline. We will demonstrate mapping of cell types and cell states to brightfield histology images on the same section from which the molecular data are generated, as well as on the independent adjacent section via registration, and finally on an independent validation tissue section. We will subsequently explore application of this approach to other HuBMAP organs including lymph node, skin, liver and lung. We will also develop 3D scalable graphics of cell types being detected using our pipeline, with a goal to develop ontological framework integrating atoms to anatomy for an objective understanding of variability in reference human atlas. We will create synergies with other HIVE teams to integrate the developed pipelines, tools with HuBMAP web-cloud portal as an easy-to-use, plug-and-play end-user plugin that is openly accessible to quantify cell counts, types, features, as well as states via uploading brightfield histology tissue images to the portal.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT The prevalence of Alzheimer’s disease and related dementias (ADRD) is expected to increase four-fold by 2050. Consequently, there are efforts to identify “actionable” risk factors, that if identified and addressed early, have the potential to prevent cognitive decline and onset of ADRD. Obstructive sleep apnea (OSA) is associated with risk of cognitive decline and ADRD, particularly among older adults. As effective treatments are available for the condition (e.g., Continuous Positive Airway Pressure [CPAP]) that are demonstrated to be highly efficacious when used adherently, OSA may be an ideal target for ADRD risk reduction. In this study, we propose to characterize associations between OSA treatment (specifically CPAP) and cognitive decline and ADRD over up to 14 years of follow-up (2010-2024) in two nationally representative NIA-funded cohorts of U.S. older adults: the Health and Retirement Study (HRS), and National Health and Aging Trends Study (NHATS). We also intend to determine whether the effects of OSA treatment on cognition differ in those at higher risk for ADRD (e.g., minority race/ethnicity, advanced age, cardiovascular risk factors/disease, etc.) in order to identify populations among whom treatment is most likely to yield effects and ways OSA treatments could be optimized in groups not benefiting. Both HRS and NHATS include nationally representative samples, have repeated performance- based cognitive measures to characterize cognitive performance trajectories and ADRD, and linkages to Medicare claims for ascertainment of OSA diagnosis and treatment. The study will use innovative methods to harmonize datasets and will address three specific aims. Aim 1 will be to determine the association of OSA with cognitive trajectories and incident ADRD from 2010-2024, and examine whether OSA interacts with other ADRD risk factors with regard to cognitive decline and ADRD risk. Aim 2 will be to determine, among individuals with OSA, whether CPAP is associated with better cognitive outcomes from 2010-2024. Specifically, we will examine whether a) receipt of CPAP (compared to no treatment) is associated with slower cognitive decline and lower risk for ADRD, b) among those treated whether initiation of CPAP is associated with a slowing of cognitive decline and lower risk for ADRD before and after initiation, and c) whether these associations vary across levels of CPAP adherence. Finally, Aim 3 will examine whether associations found in Aim 2 will differ across known ADRD risk factors. If we find that addressing OSA is particularly effective in slowing cognitive decline/preventing ADRD, especially in high-risk groups, then our study’s results will help us understand the means by which OSA can be treated in the population and contribute to development of programs to treat OSA in these populations at greatest risk. Further, it will inform efforts to curb the burden of ADRD in the U.S. and contribute substantially to dementia prevention efforts.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY We have assembled an entirely Florida-centric collaborative research team with collective expertise in microbial natural products chemistry and pharmacology, genomics, bacterial enzymology, bioinformatics, synthetic biology, chemical synthesis, and cyanobacterial and sponge chemical ecology and phylogenetics. The team is complemented by an out-of-state expert in metagenomics and bioinformatics integration. This geographical cluster of expertise being in the state with the greatest marine biodiversity in the continental US provides a dual benefit and unique opportunity to explore systems that are likely to hold some of the most promise in terms of biosynthetic potential: marine cyanobacterial communities, consisting of benthic filamentous cyanobacteria that are associated with unique microbial diversity, and sponges and their associated rich and unique microbiome in a local hotspot of biodiversity. Compounds produced by these communities are known to cover therapeutically relevant chemical space and are therefore suited as starting points for drug discovery. In a targeted fashion, we will obtain high quality (meta)genome and (meta)transcriptome sequence information from sponge-associated microbiomes and cyanobacteria using state-of-the-art sequencing techniques. We will build an integrated, multi- component platform that leverages existing bioinformatics tools and newly developed artificial intelligence-based tools to shine new light at their genomes with the goal of identifying novel biosynthetic gene clusters, particularly those unattainable with current tools, and even chemical skeletons. We will express natural products encoded by the clusters by employing five types of complementary synthetic biology systems that we have strategically developed over the past several years. These systems originating from five bacterial phyla commonly associated with both marine cyanobacterial and sponge samples cover diverse genetic backgrounds and are expected to effectively translate the identified genetic information of a variety of organisms into chemicals with proper system optimization. We will evaluate and analyze metabolites and expression profiles using LC-MS-based metabolomics and NMR and characterize associated new enzymology. Natural products derived from chemical extract and fraction libraries and those generated through our expression systems or chemical synthesis will be tested in our multidimensional screening platform, consisting of unbiased phenotypic assays in various in vitro and in vivo models as well as experimental and computational target-based functional assays. This approach will enable us to capture a broad array of activities from expressed and unexpressed genes. Selected bioactive natural products will be scaled by chemical synthesis, and bioprobes and enzyme substrates will be prepared for in-depth biological studies and enzymology research, respectively. Successful completion of these aims by our established multidisciplinary investigator team should deliver promising therapeutically important drug leads and tool compounds through thoroughly exploring marine organisms while addressing the current major limitations of natural products drug discovery over the next ten years.

NIH Research Projects · FY 2026 · 2022-08

Abstract Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive impairments, and behavioral disorders. 6.2 million people aged 65 and older are living with AD in the United States in 2021. Earlier diagnosis of AD holds particular significance as therapies are most effective during the pre-symptomatic stages before irreversible brain damage has occurred. Tau neurofibrillary tangles (NFTs), accumulating decades before symptomatic onset, can indicate the pre-symptomatic stages. According to Braak staging, tau NFTs start from transentorhinal, then spreading to hippocampus and other cortices at later stages. Detecting tau NFTs during early stages and clearly resolving their patterns is essential for early diagnosis and treatment monitoring of AD. With recent breakthroughs in tau tracer developments, Positron Emission Tomography (PET) can detect accumulation of tau NFTs in vivo. However, due to signal-to-noise ratio (SNR) and resolution limits of PET, accurate recovery of tau retention patterns in thin cortical regions is difficult. This is especially true for early stages when tau signal is weak. Additionally, recent longitudinal studies show that the accumulation change of tau deposits detected by PET is around 3 to 6 % per year for the AD group, and less for the preclinical AD group. This small annual change further challenges the signal detectability of current PET systems. Furthermore, 18F-MK-6240 is a newly developed tau tracer with higher affinity to tau NFTs and no off- target bindings near early Braak-staging regions, which makes it highly promising for early AD diagnosis. However, one issue with 18F-MK-6240 is the off-target bindings in the meninges. Given the thin nature of the cortical ribbon and its proximity to the meninges, quantitative accuracy of tau accumulation is significantly compromised. Consequently, there are unmet needs to further improve PET resolution and SNR for tau imaging. This grant application proposes deep learning (DL)-based image reconstruction methods that can improve the resolution and signal-to-noise ratio (SNR) of tau imaging. The four specific aims of this proposal are (1) to develop DL-based static PET image reconstruction; (2) to develop DL-based image reconstruction for dynamic PET; (3) to develop frameworks that can rapidly produce high-quality parametric images; and (4) to apply the proposed frameworks to 18F-MK-6240 imaging datasets. We expect the integrated outcome of the specific aims will be robust and clinically effective frameworks that can generate static and parametric images with improved resolution and SNR from static and simplified dynamic tau PET imaging.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Amputation and mortality rates are extraordinarily high for patients living with both advanced peripheral arterial disease, or critical limb ischemia (CLI), and end-stage renal disease (ESRD). Treatment choices for patients with CLI include catheter-based procedures (e.g. vascular stenting), surgery (e.g. bypass), primary amputation, or symptom control alone (e.g. pain management and wound care). Overall, CLI has dismal outcomes with a 25% mortality and a 30% major amputation rate at one year from diagnosis. Limited data suggest even worse outcomes for patients with ESRD – for example, approximately 50% mortality at 18 months. The existing literature on outcomes is limited by small sample sizes, short follow-up, and a focus on only two of the treatment options, catheter-based interventions and surgery. Patients with ESRD and CLI are often faced with difficult treatment decisions that are complicated by a lack of 1) robust real-world evidence on outcomes and 2) clinical care guidelines. Shared decision-making often relies on these types of information. ESRD is a life-limiting disease and treatment of CLI is resource-intensive. Nevertheless, there are no existing cardiovascular or nephrology society guidelines regarding CLI treatment in patients with ESRD. Knowledge of outcomes of CLI treatment in patients with ESRD, factors affecting CLI treatment selection, and guidelines to direct care would unequivocally improve care by helping to align patient values and preferences with treatment decisions. Owing to these gaps in knowledge, I propose a mixed methods approach using the national Medicare data 2017-2019 to understand 1) factors affecting treatment selection, and 2) traditional and patient-centered outcomes after treatment of CLI in patients with ESRD, complemented by 3) a Delphi panel of expert health care providers and patients to help inform the establishment of care guidelines and identification of research priorities. In addition to helping develop critical data on patients with ESRD and CLI, this proposal will be an essential step in my development as an independent investigator. My long-term career goals are to develop a research program focusing on outcomes of treatment and decision-making surrounding peripheral arterial disease. I hope to advance the field of vascular surgery health services research to improve patient outcomes and aid physicians and patients in aligning decision-making with patient goals and values. This proposal will immediately contribute to my short-term career objectives: increasing expertise with use of Medicare fee-for- service claims data, understanding the practice of nephrology as it applies to ESRD and comorbid conditions such as CLI, learning relevant advanced quantitative and qualitative techniques, and improving grant writing, leadership, and other career development skills. This proposal will take place in the resource-rich environment of the University of Florida College of Medicine under the guidance of an expert mentoring team.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Cachexia is characterized by progressive skeletal muscle and body weight loss and affects up to 80% of cancer patients. Since this loss of muscle mass contributes to weakness, reduced tolerance to conventional treatments, and increased mortality, understanding the mechanisms that drive muscle wasting is critical to the development of treatments to improve quality of life and enhance survival of cancer patients. However, in exploring the mechanisms that may drive cancer-induced atrophy of myofibers, it is important to do so in the context of the broader muscle pathologies that we and others have shown in the muscle of cachectic tumor bearing hosts, including tissue damage, non-resolute inflammation, impaired regeneration, and increased fat, collagen and calcium deposition. Unpublished proteomics data from our lab collected in the skeletal muscle of cachectic pancreatic cancer patients and, subsequently, cachectic mice bearing pancreatic tumors, releaved an enrichment of multiple pathways of the complement (Cp) system. Further immunohistochemical analyses revealed increased deposition of the central component of the Cp system, C3, and the terminal pathway/membrane attack complex (MAC) within muscle tissues of people and mice with pancreatic tumors, compared to controls. Based on these findings and the established roles of Cp proteins in causing inflammation and tissue damage, we injected mouse pancreatic cancer (KPC) cells into the pancreas of C3 knockout (C3-/-) mice and found significant protection against KPC-induced muscle wasting and weakness, that was further linked to reduced leukocyte infiltration into muscle and reduced fibrotic remodeling. These overall findings establish the requirement of Cp activation for the development of cachexia, with strong translational relevance. Aim 1 will build on these foundational findings and identify the specific Cp activation pathway and effector mechanism(s) required for the development of tumor-induced muscle pathologies and cachexia. This will reveal optimum points in the Cp pathway for pharmacological blockade. Aim 2 will utilize mouse Cp inhibitors that function at different points in the Cp pathway, targeted to sites of Cp deposition, to identify the most effective therapeutic strategy to prevent and reverse cachexia in tumor bearing mice using both the KPC model and C26 adenocarcinoma model. Aim 3 will determine the sufficiency and requirement of local myofiber-derived C3 in pancreatic cancer-induced immune cell infiltration into muscle, muscle damage, atrophy and weakness. This mechanistic aim is important because the role of local myofiber-derived Cp in muscle health and disease is almost completely unknown. Therefore, our findings here will provide mechanistic insights that will enable us to optimize and develop novel Cp inhibitory strategies for the treatment of a broad range of muscle conditions.

NIH Research Projects · FY 2025 · 2022-08

The pathogenesis and natural history of type 1 diabetes (T1D) and type 2 diabetes (T2D) are fundamentally different, but the two diseases result in many common long-term complications. Most notably, cardiovascular disease (CVD) is the leading cause of death for individuals with diabetes, resulting in a shortened life expectancy. While rigorous blood glucose management reduces the risk for CVD development, the vast majority of diabetes patients are unable to meet recommended HbA1c targets. Given the high prevalence of diabetes (10.5%) and pre-diabetes (33%) in the United States (U.S.), it is imperative to understand diabetes-related CVD pathogenesis to support the development of optimal intervention and treatment strategies. However, studies comparing CVD mechanisms in T1D versus T2D are critically lacking. To address this, we propose to establish a Cardiovascular Repository-Type 1 Diabetes (CARE-T1D) program to facilitate collaboration and multi-modal data acquisition across a large network of investigators. Through our leadership of 6 organ procurement and biospecimen sharing research programs, we have 15 consecutive years of operational experience and well-established programmatic infrastructure for collecting and distributing 16 different types of transplant-quality tissue from human organ donors, including a recently concluded kidney project and current heart pilot program. We will leverage our productive relationships with all 57 U.S. Organ Procurement Organizations, centralized 24/7/365 Call Center and Organ Processing and Pathology Core to procure, to swiftly process and bank a complete CVD-related tissue panel (heart, kidney, vasculature, blood) from 60 donors with CVD, evenly distributed across three groups (T1D, T2D, age/sex-matched no-diabetes controls). Following whole organ radiology and calcium scoring, anatomical dissection will be systematically performed by our highly experienced staff to prepare biospecimens in a variety of formats (e.g., FFPE blocks, OCT blocks, flash-frozen), with protocols evolving to support emerging needs for research applications. Each case will be subjected to tissue-specific stains with histopathologic examination by board certified pathologists and QA/QC analysis. Resulting data will be made available alongside de-identified donor information and medical records in a secure searchable Data Portal to aid investigators in selecting sample sets for their research. We propose to establish a Scientific Advisory Board to evaluate research proposals and sample requests, modeled after our existing Tissue Prioritization Committee. We will distribute biosamples to approved researchers seeking to apply multimodal approaches for deep phenotyping of specimens to study CVD progression in T1D vs T2D. The Data Portal will also support visualization and sharing of all externally generated data types. Finally, we will organize annual meetings to promote collaboration across the Cardiovascular Biorepository Consortium. In sum, we expect the proposed CARE-T1D program will support discovery and mechanistic research, conducted by a collaborative network of investigators, that will increase our understanding of CVD in diabetes, leading to early detection as well as novel treatments specific for both T1D and T2D.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Since 2007, more than 40 glucose-lowering drugs (GLDs) have been approved by the US Food and Drug Administration to treat diabetes. These newer GLDs have been proven to have higher cardiorenal benefits than older classes when applied in people at high risk of cardiovascular and kidney disease. However, the introduction of these high-cost GLDs has led to significant quality and equity concerns in diabetes care: socially disadvantaged individuals tend to have limited access to newer GLDs due to barriers related to social attributes (e.g., income, education), resulting in gaps and disparities in achieving optimal health outcomes. There is, therefore, an urgent need to improve the quality of care and equity in using newer GLDs among millions of Americans living with type 2 diabetes (T2D). Previous studies have found that programs that improve the quality of care by promoting treatment in targeted clinically high-benefit user groups lead to equity improvement because high-benefit users from socially disadvantaged subgroups often have larger gaps in care thus benefit more from these programs. However, critical knowledge gaps exist in identifying the clinically high-benefit users of newer GLDs and designing policy- level interventions that can adequately motivate patients’ newer GLDs use while having good long-term health and economic outcomes. Thus, the OBJECTIVE of this proposed project is to identify clinically high-benefit T2D patient subgroups for newer GLDs and generate empirical economic evidence for designing policy-level interventions to improve the quality of care and health equity in T2D care. High-quality comparative effectiveness research (CER) requires the patients to have complete data records which can track event encounters and treatment exposure with high accuracy. These individuals were often referred to as “loyal patients.” In this proposed project, we will develop a computable phenotype (CP) for “loyal patients” using OneFlorida EHRs and cross-network validate the CP using REACHnet EHRs (Aim 1). To identify clinically high-benefit T2D patient subgroups for newer GLDs, we will conduct comparative effectiveness and safety analyses of newer GLDs versus guideline-recommended alternatives across patient subgroups using rigorous causal inference methods and a machine-learning (ML) approach. The high-benefit T2D patient subgroups will be identified using EHRs of “loyal patients” from OneFlorida and cross-validated in REACHnet (Aim 2). At last, we will evaluate the impact of potential policy-level interventions for promoting newer GLDs use in high-benefit users on health, economics, and equity outcomes. Leveraging an advanced ML algorithm developed by PI, we will also identify the ideal cost-sharing structure at a health-plan level to maximize drug adherence while reducing the payers' burden. The proposed research is significant because it will provide solutions for an emergent public health issue in quality of care and health equity in diabetes management. This study is innovative because we will use cutting- edge machine-learning methods, simulation models, instrumental variables, and two of the largest PCORnet EHRs to tackle a challenging and innovative research question.

NIH Research Projects · FY 2025 · 2022-07

Modified Project Summary/Abstract Section The United States (US) is the most affected country worldwide by the ongoing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV 2) pandemic. By the beginning of 2021, incidence of severe coronavirus disease 2019 (COVID 19) cases, hospitalization burden, and deaths appeared to be decreasing. Unfortunately, the emergence of new, highly transmissible variants of concern (VOCs), such as the Delta variant that rapidly became dominant in the US, caused a renewed epidemic surge in mid 2021 and continues to pose challenges for disease control. Although evidence on increased mortality and worse clinical outcomes among people with HIV (PWH) infected with SARS CoV 2 remains mixed, several studies suggest that PWH may have a higher likelihood of severe disease or death compared with individuals without immune dysfunction. While most individuals clear SARS CoV 2 infection within 2–4 weeks, persistent infection in immunosuppressed individuals has been associated with intra host emergence of multi mutational viral variants, including mutations at sites linked to immune evasion. The overarching goal of this project is to investigate SARS CoV 2 intra host genomic evolution in the context of HIV infection by developing a phylodynamic and artificial intelligence framework (PhAI CoV) to assess the emergence and likelihood of SARS CoV 2 variants of concern in immunocompromised PWH. We hypothesize that SARS CoV 2 infection in PWH can result in enhanced viral evolution that can be efficiently characterized using phylodynamic methods and predicted using artificial intelligence algorithms.

NIH Research Projects · FY 2025 · 2022-07

SUMMARY The American Burn Association estimates that there are ~3,500 deaths each year from burn injuries. There are multiple influences on morbidity and mortality in burn patients, with inhalation injury among the most significant as it leads to increased susceptibility to opportunistic bacterial infections and the associated morbidity and mortality. A trifecta of clinical need is associated with this clinical problem: 1) we lack the ability to predict risk of infection, 2) we do not understand the mechanism of infectious risk, and 3) we are unable to restore a patient’s immune system to homeostasis after injury to enable adequate control of infectious agents. The overall objective of this application is to delineate mechanisms responsible for the cycle of uncontrolled inflammation following burn-injury to refine prediction models patient outcomes and to refine therapeutic approaches to restore immune homeostasis, thus decreasing susceptibility to infection and preventing the associated morbidity and mortality. We and others have demonstrated in human samples and mouse models that burn and burn + inhalation (B+I) injury generates the local and systemic release of numerous Damage-Associated Molecular Patterns (DAMPs). DAMPs promote interactions, via key immune regulators, such as mammalian Target of Rapamycin (mTOR) to induce reactive oxygen species (ROS), inflammatory cytokines, and chemokines which results in tissue damage and immune cell recruitment. Immune homeostasis is normally restored at least in part by the transcription factor Nuclear Factor-Erythroid-2-Related Factor (NRF2). Our preliminary data demonstrate that Nrf2-/- knockout mice have profound mortality after B+I injury. However, our preliminary data also demonstrate that while pulmonary immune cell NRF2 protein translation is rapidly increased after B+I in wildtype mice, it is not translocated to the nucleus. Thus, we hypothesize that the NRF2-mediated homeostasis following burn and B+I injury is insufficient, but that pharmacological activation of the NRF2 pathway has the potential to reduce acute immune dysfunction. Using our pre-clinical models of burn and B+I injury, we will define NRF2-specific mechanisms of acute immune dysfunction following burn or B+I injury and validate these findings in human cohorts within in our high-volume burn center. In addition, we will utilize microparticle technology to develop and characterize NRF2-driven therapy to improve post-injury immune dysfunction. As we appreciate that the response to burn and B+I is multifactorial, we will leverage this technology to combine NRF2 activation with a second approach and inhibit mTOR to provide a novel multimodal therapeutic approach. The efficacy of these approaches will be evaluated using our pre- clinical models of burn and B+I. We are uniquely poised to successful complete this proposal which will allow us to fill the existing knowledge gaps and improve long-term outcomes of burn and B+I patients.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are complex neurodegenerative diseases that affect motor neurons in various regions of brain and spinal cord with devastating impacts on a patient’s health and lifespan. While research has identified ALS and FTD mutations in a number of genes (i.e. C9orf72, MAPT, SOD, and GRN), approximately 90% of ALS and 60% of FTD patients present as sporadic cases (sALS & sFTD) with unknown genetic etiology. Complex disease mechanisms coupled with a large genetically and phenotypically heterogeneous patient population have severely limited research and therapeutic success for these diseases. The discovery of the intronic C9orf72 G4C2 repeat expansion mutation as the most common genetic cause of ALS and FTD links these diseases to the larger family of microsatellite expansion disorders. Amongst these diseases is spinal-bulbar muscular atrophy (SBMA), a CAG•CTG disease that, like ALS and FTD, also affects motor neurons. A growing number of expansion disorders are reported to express proteins in multiple reading frames by repeat associated non-AUG (RAN) translation. Our recent unpublished findings show that novel polySer and polyLeu RAN proteins accumulate in at least six of the ten CAG•CTG polyGln diseases. These observations raise the possibility that novel polySer and polyLeu RAN proteins accumulate in the spinal-bulbar muscular atrophy (SBMA) and that other unidentified RAN proteins may contribute to sALS and sFTD. Our central hypothesis is that repeat expansion mutations that express novel RAN proteins substantially contribute to sALS, sFTD and spinobulbar muscular atrophy (SBMA) and that therapeutic approaches that reduce RAN protein levels will improve disease in preclinical models. To address this hypothesis, we have developed an innovative pathology-to-genetics strategy that enables rapid and direct identification of novel RAN protein producing expansion mutations from patient DNA. We are excited to report that in an initial screen, ~30% of sALS autopsy cases of unknown genetic etiology (i.e. C9 and SCA36 negative) were positive for GR or PR RAN protein aggregates suggesting the presence of novel expansion mutations. In this proposal, we will test the hypothesis that novel types of RAN proteins contribute to sALS, sFTD and SBMA (Aim 1) using immunoassays and patient blood and autopsy tissue samples. In Aim 2, we will utilize an innovative dCas9READ method to identify novel repeat expansion mutations in RAN(+) sALS and sFTD cases and study the toxic effects of putative disease-causing expansion mutations. Lastly, we will test the hypothesis that decreasing RAN translation using AAV-PKR(K296R) or metformin will improve disease phenotypes in patient derived induced models and mouse models of sALS, sFTD and SBMA (Aim 3). Taken together, these studies will provide critical insights into the molecular mechanisms of sALS, sFTD and SBMA and facilitate the development of unifying therapeutic approaches to fight these devastating diseases.

NIH Research Projects · FY 2025 · 2022-07

The All of Us University of Florida Program works with the All of Us NIH team towards Program Strategic Goal #4: Researchers on the Workbench. Under this Goal, in year 4 we are pursuing five milestones. Firstly, we will test the All of Us Curriculum that our team developed in Year 3 to cover the three tiers (Public, Registered and Controlled) and Researcher Workbench features, along with specific medical condition Use Cases of concern to communities in the United States: Cancer, Depression, Diabetes, Hypertension, and the Consequences of Cannabis Use. Next, we will evaluate and refine the All of Us Curriculum with the assistance of other All of Us partner sites and the Association for Clinical and Translational Science volunteers from across the country. Additionally, we will assist the NIH as subject matter experts with the curriculum which may require reviewing new data types, features, and functionalities within the Researcher Workbench, providing feedback on emerging initiatives, and pilot testing new products for researchers. We will continue to work closely with our NIH Program Officer and funded partners within the timeline to maximize the full potential of the All of Us Research Program and Researcher Workbench to advance precision medicine.

NIH Research Projects · FY 2025 · 2022-07

Abstract: Lung adenocarcinoma (LUAD) is the major histological subtype of lung cancer and the leading cause of cancer-related mortalities worldwide. For a substantial number of LUAD patients, the only treatment options available are traditional multi-agent chemotherapy coupled with surgery and/or radiation. For these patients, the 5-year survival remains disappointingly low. Additional molecular mechanisms driving LUAD proliferation and tumorigenesis remain a critical knowledge gap in lung cancer research, and a major barrier for the development of personalized therapies. Recent reports, including our own, reveal critical roles for glycogen in lung tumor progression. Building on these foundational studies, we developed a robust and precision technology to visualize glycogen in situ with 50 µm spatial resolution using mass spectrometry imaging that provides 1,000x increased sensitivity compared to previous methods. Using this technology, we defined glycogen levels in 122 NSCLC patients treated at the University of Kentucky’s NCI Designated Cancer Center. Our preliminary data demonstrate that: 1) significantly elevated glycogen is observed in LUAD and not in normal lung tissue. 2) Elevated glycogen is a LUAD tissue-specific hallmark and is not observed in lung squamous cell carcinoma. 3) LUAD-glycogen is structurally unique with increased phosphorylation and branching. 4) This LUAD phenotype correlated with marked protein decreases in the glycogen phosphatase laforin. Strikingly, laforin knockout in model lung cell lines and the KrasG12D/p53-/- LUAD mouse model drives: 1) glycogen hyper-phosphorylation, 2) increased affinity with the master metabolic regulator AMP-activated protein kinase (AMPK), 3) decreased AMPK activity, and 4) accelerated tumor proliferation and progression. We hypothesize that the structurally unique LUAD-glycogen is a critical component of LUAD metabolism, proliferation, and progression. The overall objective of this study is to define the etiology of LUAD-glycogen on both cancer metabolism and tumor progression. To achieve this, we will: Define the LUAD-glycogen clinical course and its interaction with AMPK (Aim 1). Then, we will define the signaling role of LUAD-glycogen in cellular metabolism through AMPK (Aim 2). Finally, we will establish the role of LUAD-glycogen in tumor progression and early transformation in vivo (Aim3). This proposal builds on exciting and rigorous preliminary data and presents an integrated approach to define this unique LUAD hallmark of excess glycogen utilizing robust, complementary, and state-of-the-art methodologies such as mass spectrometry imaging, protein and glycogen biochemistry, and targeted metabolomics. The salient findings from this proposal will significantly advance the knowledge base regarding the roles of glycogen in LUAD biology and progression and drive the discovery of personalized therapies that can be leveraged for the LUAD population that only qualify for conventional chemotherapy.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT The design and implementation of improved therapies for vascular diseases are dependent on a better understanding of the basic physiological processes involved. Such processes are biologically complex, with elements at the molecular, cellular, organ system, and integrative levels. Research into the pathophysiology of vascular diseases and their treatments will be strengthened by training vascular surgeon-scientists in investigative methods allowing them to develop and maintain state-of-the-art laboratory skills and pursue novel strategies for cure and prevention. The goal of the University of Florida - Interdisciplinary Training for Vascular Surgeon Scientists (ITVSS) Program is to provide the training required for success as future academic surgeons and scientists in the field of vascular surgery. Graduates of our training program will be prepared to join an academic surgical faculty, conduct independent research, compete for grant funding, and provide translational surgical expertise at their institutions. At the core of our program are the following specific aims: 1) provide a 2-year integrated training program for four surgery residents and/or fellows to create a career pathway for conducting mechanistic and clinically relevant translational research in vascular disease; 2) implement a training program that promotes a learning cycle of concrete experiences, active experimentation, reflective observation, and abstract conceptualization; 3) closely monitor and track trainee-related experiences and outcomes that will serve as a foundation for evaluating the current status of the program and for making continuous quality improvements; 4) create a culture for professional excellence based on enhancing rigor, reproducibility and transparency in research and establishing a career trajectory as leaders in the surgical delivery of vascular care; and 5) maintain the research-focused, career trajectory developed during the fellowship by promoting a culture of scholarly activity, foster interaction among the ITVSS trainees, mentors, and Directors during the completion of clinical training. Unique aspects of the ITVSS Program include: 1) a high-density of NIH-funded surgeon-scientists providing the necessary training environment and role models; 2) use of a team mentoring approach, where each trainee has an identified research and clinical mentor; 3) trainee engagement in an “Immersive Training Experience” that is outside of their research focus area, to provide the foundation for conducting team-based, multi- disciplinary research; 4) support by a robust vascular research portfolio that offers trainees high-quality experiences in basic, translational, outcomes, and clinical research; and 5) institutional backing that provides administrative and supplemental financial resources to maximize the training experience.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract Given the paucity of effective treatments for TMD and the overuse of pain medication, well-designed studies are needed to evaluate non-pharmacological alternatives to treat this common and disabling chronic pain condition. Our goal in this study is to conduct a double-blind, sham-controlled, randomized clinical trial of multimodal Photobiomodulation (PBM) for TMD pain. Also, we propose to determine if PBM-induced changes in inflammation and pain sensitivity contribute to PBM’s analgesic effects. A total of 130 TMD participants will be recruited through community-based advertisements. Participants will complete a computer-assisted telephone screening (CATI). Eligible participants will be age 18 and older with pain intensity of ≥30 on a visual analog scale (0-100). Participants will be excluded if: a) starting a new daily prescription medication for the management of pain within 30 days before CATI; b) use of injection therapy (e.g., tender or trigger point injections, steroid injections) for the management of pain within 2 weeks before the CATI; c) starting occlusal appliance therapy within 30 days before CATI; d) history of facial trauma or orofacial surgery within 6 weeks before CATI; e) active orthodontic treatment; f), psychiatric hospitalization within one year before the screening. Participants eligible after CATI will be scheduled for a pre-randomization visit (V0), eight treatment visits (V1 to V8), one post- treatment visit (V9), an internet/ phone follow-up at 1 and 3 months, and six-month follow-up clinical visit (V10). V0 will include informed consent, completion of a detailed medical history and to assess clinical pain and interference, participants will complete the Pain, Enjoyment, General Activity (PEG) Scale followed by a clinical exam to confirm TMD status according to the Diagnostic Criteria for TMD (DC/TMD), Pressure Pain Sensitivity (PPT) and blood draw. V1 will be the randomization visit to either receive PBM or Placebo. V2-V8 are treatment visits. V5 is also the Midway visit, therefore, a blood draw, DC/TMD exam and PPT will be performed before treatment. At V9 (post-treatment visit), post-treatment outcomes will be assessed, PEG, DC/TMD exam, PPT and a blood draw. PBM/Placebo treatment: We will use three types of active/Placebo probes; A) Single Laser 808 nm, 4.75W/cm 2; B) Laser Cluster, five x 810 nm, 5.96W/cm 2 and four LED 660nm, 0.05W/cm 2and; C) LED Cluster, 56 X 660nm 0.51W/cm2 and 48 850nm, 0.45W/cm2, 950mW total applied to multiple craniofacial sites. Analyses will determine treatment effects on the primary outcome (pain intensity) and multiple secondary outcomes and will examine, exploratorily, whether changes in inflammation and pain sensitivity mediate treatment response. Findings from this rigorously designed trial will provide the most definitive evidence to date regarding the effectiveness and mechanisms of PBM for treating TMD pain.

NIH Research Projects · FY 2025 · 2022-07

This is a renewal application for the CDC Southeastern Center of Excellence in Vector-Borne Diseases: Gateway Program (SECVBD) led by the University of Florida with both well-established and burgeoning collaborations with 13 universities with strong vector biology programs across the Southeastern United States (U.S.) and Puerto Rico. This integrated academic network carries the full and integrated support of state departments of health, state and federal agricultural agencies, U.S. military programs, national and international associations, and >60 local mosquito and vector control programs. Our proposal leverages the expertise across a broad remit of basic and applied vector biology as well as epidemiological disciplines to continue to maintain the success of our program in (i) establishing an integrated community of practice in public health entomology consisting of academic institutions throughout the Southeast and local, state, and federal public health agencies, to facilitate existing and future efforts in vector-borne disease (VBD) surveillance and control and (ii) expanding an effective academic, online, and internship-based training program in basic public health entomology to augment the cohort of personnel who are trained with the requisite knowledge and skills to quickly detect and respond to VBDs across the U.S., and (iii) initiating the drive towards an evidence-based set of recommendations and a tailored template of a “surveillance-response program” to ensure that local mosquito and vector control associations can better monitor and control VBDs in the U.S. For the next iteration of the SECVBD, we take advantage of the biocomplexity of tick- and mosquito-borne diseases in the Southeastern U.S. and Puerto Rico and leverage an integrated, data-driven, applied research program designed to produce maximal output in novel interventions and training paradigms within a 5-year period to tackle the following CDC priorities: (a) Laboratory/field evaluations of emerging technologies to suppress host-seeking ticks or disrupt pathogen transmission cycles; (b) Field studies aimed at optimizing application of tick control products to provide recommendations for pest control firms and homeowners regarding how to optimize tick suppression; (c) Assessments of the potential for incorporating tick management into existing mosquito management programs; (d) Investigations on the knowledge, attitudes, & behaviors regarding tick control methodologies, (e) Evaluation of the impact of existing public education programs on TBI risk & behaviors to prevent tick bite; (f) Field evaluations of emerging technologies to suppress mosquito adults (insecticide resistance); (g) Field evaluations of mosquito management approaches based on commercially available methods and products (both entomological and human-related outcome measures); and (h) Evaluations of the effectiveness of operational control activities targeting all mosquito stages (including insecticide resistance). The goal is to maintain our established, comprehensive, applied, and basic research program to further our understanding of VBD transmission to prevent VBDs in the continental U.S. and territories.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract This objective of this proposal is to develop new synthetic methods for the enantioselective synthesis of bioactive molecules. Studies are centered on utilizing a new class of atropisomeric chiral biaryl ligand being developed in our laboratory. Preliminary work has found that imidazole-based biaryl P,N-ligands excel at promoting enantioselective copper catalyzed carbon-carbon bond-forming reactions. Our goal is to capitalize on the differences in behavior between these ligands and existing biaryl ligands to enable new reaction technology for applications in discovery chemistry. The research in this application is focused on gaining a mechanistic understanding of the structural underpinnings responsible for new and unique reactivity imparted by this new ligand scaffold. Our first aim outlines plans to develop new dearomatization reactions of nitrogen heterocycles. In this aim we describe chemistry that will push the field past chiral carbocycles and single-heteroatom heterocycles to heterocycles with >1 heteroatom. Through preliminary results we demonstrate addition to pyridine, but more importantly pyrazine, pyridazine, and pyrimidine. These latter examples are unprecedented in the literature and the success of these catalytic enantioselective dearomatization reactions is developed here to provide rapid access to complex natural products like svetamycin B, saxitoxin, and batzellidine B as well as additional chiral nitrogen-containing building blocks. The second aim is focused on catalytic enantioselective addition reactions to C=N Bonds independent of nitrogen substitution. In catalytic enantioselective processes, it is common for iminium ions to require 2 identical N-substituents to avoid the E/Z issue. We have found that catalytic enantioselective alkynylation using StackPhos lifts this requirement and the use of two different N- substituents is possible. This breakthrough allows the move from bis-protected amines (e.g. N,N-dibenzyl) to the incorporation of groups needed for the synthesis. Here we capitalize on this for an expedient synthesis of kopsanone and other chiral heterocycles such as morpholines. In addition, through preliminary results, we also demonstrate that addition to imines and nitrones in high ee is possible, despite the E/Z-isomer issue. Extensive preliminary results support these aims and we predict that the chemistry developed here will be of broad impact to practitioners in academia and industrial settings, particularly the pharmaceutical and biotech sectors.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Opioid use can induce aspiration, which greatly increases the risk of pneumonia. Mortality rates of aspiration pneumonia can approach 40%. The larynx plays a pivotal role in protection of the airways by preventing ingested materials from entering the trachea. This organ participates in different airway protective behaviors, but the initial protective response to intrusion of material is the laryngeal adductor reflex (LAR). The LAR consists of rapid adduction of the vocal folds following stimulation of laryngeal sensory afferents. No information exists on the sensitivity of central pathways responsible for the LAR and their contribution to maladaptive laryngeal responses to opioids. Based on preliminary data and model simulations, we have developed the following hypothesis: opioid-sensitive circuits in the nucleus of the solitary tract and nearby reticular formation (NTS/RF) include a network of neurons with tonic expiratory (t-E) and non- breathing modulated (NBM) activity patterns that regulate reflexive laryngeal adduction through their functional interactions with cells in the ventrolateral respiratory network (VL). This project has two Specific Aims: 1) Identify the network between NTS/RF and VL neurons that regulates the LAR. 2) Determine the central effects of opioids on ipsilateral and crossed pathways in the NTS/RF that regulate coordination of motor drive, mechanics, and vocal fold movements during LAR. We anticipate this project will lead to: a) identification of critical elements of the central reflex pathway for the LAR that are sensitive to opioids, b) identification of the functional relationships for the production and regulation of the LAR: those within NTS inter-neuronal networks, and those between t-E, NBM, NTS/RF, and VL neurons, and c) a new clinically-useful neuromechanical model of vocal fold coordination that will enable prediction of the effects of depressant drugs on the airway protective actions of the larynx. This model will feature functionalities not currently available to clinicians, such as estimation of the impact of unilateral vocal fold hypotonia on laryngeal function. This new knowledge will provide a critical step in understanding the neurogenesis and neuropharmacology of the LAR and how opioids compromise airway protection.

NIH Research Projects · FY 2025 · 2022-06

Bacterial viruses, known as bacteriophages or phages, are among the most abundant biological entities on the planet. These viruses vary broadly in terms of genome content, infection mechanisms, replication cycles, and structure. With an increasing interest in using bacteriophages to combat antibiotic resistant bacteria, it is necessary to understand mechanisms of how these viruses infect their hosts; replicate and regulate both their genes and their hosts’ genes; and how they persist in various environments, including the human body and/or storage conditions. However, our current model systems inherently only represent a fraction of bacteriophage diversity. The long-term goal of this work is to develop an additional bacteriophage system—the Moogleviruses— that appear to be ubiquitous in the environment and clinically useful qualities. They have, however, only recently been isolated because they do not commonly infect the model species of bacteria Escherichia coli and Salmonella enterica. Instead, these viruses are often isolated against the pathogenic bacteria Shigella flexneri or opportunistic pathogens such as Citrobacter freundii. Because these viruses are obligately lytic and specific to one of the most common etiological agents of diarrhea, S. flexneri, they could be used for controlling this species of bacteria in food or water, or for treating antibiotic-resistant infections. At this time, however, we lack sufficient understanding of their biological processes. Moogleviruses have distinct characteristics versus other bacteriophages, including a semi-specific host range, a large number of tRNAs encoded in their genomes, and uncommon capsid and genome sizes. The central hypothesis of this work is that these viruses use alternative strategies to infect and persist compared to more thoroughly characterized model systems. The objectives of this specific proposal are therefore to determine the mechanisms Shigella-infecting Moogleviruses use to identify their hosts, modulate the expression of viral and host genes on a translational level, and assemble into new particles that can persist in the environment. The expected outcomes of this proposal are: 1) the identification of critical interacting regions between the phage receptor-binding proteins and both primary and secondary receptors on the S. flexneri host, along with determining the kinetics of attachment and entry; 2) complete Ribo- seq and RNA-seq datasets from a variety of environments and infection conditions, leading to a mechanistic understanding of how phage infection alters translational efficiency of phage and host genes; and 3) a broadly resolved assembly pathway of the Mooglevirus capsid, with an indication of proteins or protein domains responsible for affecting capsid stability. This work will have impacts for both basic biology, expanding our repertoire of known bacteriophage strategies and mechanisms for infection and persistence; and medical application, informing how these phages themselves could be applied to combat S. flexneri infections in the clinic, or how their properties could be used to engineer novel phages for medical or industrial applications.