University Of Florida

NIH Research Projects · FY 2026 · 2021-11

Abstract Our central premise is that HIV transcriptional inhibitors can be used as latency promoting agents (LPAs) in block-and-lock functional cure approaches, aimed at reducing residual viremia during antiretroviral therapy (ART) and limiting viral rebound upon treatment interruption (TI). This hypothesis came about with the discovery of the Tat inhibitor, didehydro-Cortistatin A (dCA). In in vitro and in vivo models of HIV latency combining ART with dCA accelerated HIV-1 suppression to below the limit of detection and blocked viral rebound upon TI or stimulation with latency reversing agents (LRAs). The transcriptional shutdown by dCA resulted in the heterochromatinization and loss of RNAPII at the HIV promoter. We recently reported on another LPA, the generic drug Spironolactone (SP). Interestingly this drug degrades the host XPB subunit of the general transcription factor TFIIH inhibiting HIV transcription in primary cells from infected individuals and blocking viral reactivation from latency without affecting cellular transcriptomics7. This study is important because it highlights that a host factor can be targeted to silence HIV without affecting cellular viability. In sum, the more knowledge we gain on the interconnectivity between Tat, transcription factors regulatory mechanisms and how these affect nucleosomes positioning around the HIV promoter, the more we can leverage transcriptional regulators as antiviral targets. To identify novel host factors regulating HIV transcription we used a chromatin affinity purification approach together with mass spectrometry (ChAP-MS). We identified p32 (ASF/SF2 splicing factor-associated protein) and FUBP3 (Far upstream element binding protein 3) in productively infected ART treated promoters but not in dCA-treated where HIV was silenced; and PHB2 (prohibitin-2) was enriched in dCA treated promoters but reduced in ART only treated ones. These results suggest that p32 and FUBP3 are HIV transcriptional activators while PHB2 is a HIV silencing factor. Preliminary studies confirmed their transcriptional activity on HIV. Here we propose a detailed molecular mechanistic study on how p32 and FUBP3 enhance and how PHB2 inhibits HIV transcription. Understanding their role in pre-initiation complex (PIC) formation, initiation, elongation and nucleosome organization at the latent HIV promoter will be key for developing successful strategies for durable HIV silencing. Strong effects of one or more of these factors will prompt the search for small-molecule modulators. Our overarching goal is to durably silence latent HIV proviruses by driving each into stable heterochromatin where they will remain “locked.” We propose the following aims: Aim 1: Define the roles of p32, FUBP3 and PHB2 in HIV transcription and chromatin structure. Aim 2: Identify protein complexes associated with p32, FUBP3 and PHB2 and study interaction with Tat. Aim 3: Investigate the “block-and-lock” approach using single or combinations of factors and their dependence on Tat/TAR circuitry.

NIH Research Projects · FY 2025 · 2021-11

PROJECT SUMMARY A single toxicant exposure during development can produce reproductive defects in adulthood and subsequent generations, presenting a major hurdle in the prevention and treatment of human infertility. Despite its significance, however, the mechanisms that mediate this process are poorly understood. Endocrine disrupting chemicals (EDCs) play a role in the increasing incidence of male infertility worldwide, and mounting evidence suggests that EDC exposure can alter gene expression and the epigenome. Our long-term goal is to determine how environmental toxicants interfere with reproductive health so that evidence-based strategies to prevent and treat adult-onset and transgenerational disease can be developed.!The overall objective for this NIEHS R01 Award (PA-19-056) application is to determine genome function alterations and epigenetic regulation of environmentally-influenced infertility. The central hypothesis is that sublethal EDC exposure during male gonad development leads to genomic and epigenetic dysregulation that alters testicular mitochondrial function in exposed generation and subsequent generations. The rationale for the proposed research is that investigation of the mechanisms underlying EDC induced infertility will advance prevention, risk-assessment, diagnostic, and treatment strategies for human male infertility. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine testicular cell-type specific and life stage specific changes in genome function to identify critical windows for biomarkers of effect and gene relationships; 2) Identify changes in the epigenome related to phenotypic and genetic endpoints; 3) Determine multigenerational and transgenerational cell-specific transcriptomic and epigenetic changes induced by ancestral exposure. Ultimately, these results will identify critical windows for biomarkers of effect, inform the interplay among pathways mediating toxic endpoints.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Tourette syndrome (TS) is a continuous lifelong condition that is highly prevalent, socially disabling, and in some severe cases, physically injurious. DBS has emerged as a promising treatment option for addressing uncontrollable tics in medically resistant and severe cases of TS frequently involving self-injurious behavior. We have undertaken a major informatics initiative by establishing the International TS DBS Registry and Database, a multi-country consortium that has captured long term outcomes of 277 TS DBS patients representing 50-75% of all TS DBS cases worldwide. From these outcomes, two deep brain targets have emerged as potentially effective: the centromedian nucleus region (CM) of the thalamus, and the anterior globus pallidus internus (aGPi). However, our current understanding of tic generation is limited by many factors including a lack of animal models for TS, apparently normal brain structure on structural imaging, and the impracticality of studying involuntary motor tics with functional imaging. Next generation closed-loop DBS systems can record brain activity in patients with TS and identify the neurophysiological correlates of tics. Moreover, these devices can deliver stimulation in response to a patient's symptomatic state. Our overall goal is to develop neurophysiology driven and connectivity-guided closed-loop DBS systems for the improved treatment of TS. To this end, we will implant 8 medically resistant TS patients with bilateral leads in the CM and aGPi. In Aim 1, we will identify structural network projections from CM and aGPi to guide pre-operative surgical planning and post-operative selection of stimulation parameters. In Aim 2, we will identify neurophysiologic correlates of tic genesis in the CM and aGPi. We will also study thalamo-pallidal network interactions leading to and during tics. In Aim 3, we will test the feasibility, safety, and efficacy of closed-loop TS DBS. We expect that closed-loop stimulation will provide more effective and personalized treatment options with longer battery life and fewer adverse effects.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Lewy body dementia (LBD) is the second most common neurodegenerative dementia in the United States and one of the Alzheimer’s disease-related dementias (ADRDs). It affects 1.4 million Americans and their caregivers. Individuals with LBD are affected by both physical and cognitive impairments, and eventually require continuous supervision leading to high caregiver burden. A patient-centric specialized interdisciplinary care model, the Neuromedicine Service and Science Hub model, addresses physical, cognitive, nutritional, social support, and caregiver needs. However, this model of care is not easily accessible or offered across the country. The overarching goal of my proposal is to improve patient and caregiver health outcomes by increasing access to specialty care for individuals with LBD and their caregivers. The objective of the proposed study is to convert this in-person patient-centric interdisciplinary care model to a virtual platform using stakeholder engagement (Aim 1). After adapting the care model to virtual delivery, I will conduct a pilot randomized, controlled clinical trial including 30 individuals with LBD and their caregivers for 6 months to determine the feasibility, acceptability, and appropriateness of this intervention, as well as to obtain pilot outcome data (Aim 2). Additionally, the study will pilot the usability of a wearable sensor to objectively assess patient outcomes, including life space mobility (Aim 3). Life space mobility, a measure of the area in which someone moves through, closely correlates with quality of life, falls, and mortality. My research goal is to establish and iteratively improve a virtual interdisciplinary model of care allowing patients with LBD and their caregivers to access sub-specialty care from their home. Through this K23, I have organized an interdisciplinary mentoring committee and training plan to develop expertise in pragmatic clinical trial design, dissemination and implementation science, qualitative research methods, data analysis, and research conduct. The mentoring team consists of world-renowned experts in interdisciplinary models of care in neurodegenerative conditions (Michael Okun, Benzi Kluger), stakeholder engagement and qualitative research in LBD (Melissa Armstrong, James Galvin), dissemination and implementation science (Stephanie Staras) and wearable technology (Todd Manini). This research will be conducted at the Fixel Institute for Neurological Diseases at the University of Florida, which has the integrated telemedicine capabilities, interdisciplinary care framework, and clinical/research resources to conduct this study. The expertise and skills gained by this 5 year career development award will help accomplish my long-term career goal to become an independent investigator researching effective care delivery to individuals with LBD and their caregivers.

NIH Research Projects · FY 2025 · 2021-09

This study will establish the molecular mechanism linking mild repetitive traumatic brain injury (mrTBI) and onset of tau pathology that is associated with Alzheimer’s disease (AD). Our preliminary data suggest that endoplasmic reticulum stress is a notable and long-lasting cascade that is activated by injury. ER stress activates a protein called PERK, which is responsible for initiating protective pathways that help restore ER function. However, long- term activation of PERK leads to cell death. Brain cells are particularly susceptible to PERK-mediated cell death. Indeed, a common sign between TBI and AD is PERK hyperactivity. We recently established that another com- mon pathological hallmark of TBI and AD, abnormal aggregation of the protein tau, is driven by chronic activation of PERK. PERK induces tau to adopt toxic conformations that are associated with disease. Therefore, the overall hypothesis of this project is that TBI induces long-lasting activation of PERK, which in turn catalyzes the formation of pathological tau species. This ultimately leads to increased risk for AD. We will test our hypotheses using mouse models in two aims. In Aim 1, we will determine the conditions under which mrTBI causes activation of PERK. To accomplish this objective, mice will be subjected to mrTBI at different intensities and for different time points, and the levels of active PERK will be measured. In addition, we will determine the extent of tissue that shows PERK activity. In Aim 2, we will manipulate PERK activity in mouse models of tauopathy that have suffered mrTBI. We expect that PERK activation will cause more tau pathology and induce damage to brain function. Conversely, PERK inhibition will restore brain function and prevent tau pathology. Aim 3 will determine the va- lidity of using PERK as a biomarker of TBI. Our preliminary data suggest that individuals who suffered one or more TBIs in their lifetime have two times more active/total PERK ratio in their blood. These data support our enthusiasm to expand our studies into a much larger cohort. If successful, this grant will not only identify a molecular mechanism that links injury and AD, but it will also highlight a key pathological pathway replete with therapeutic targets. Logical extensions of these studies involve testing inhibitors of the PERK pathway for po- tential therapeutic value. It will also offer relief to the 1.7 million people in the United States who suffer a TBI annually. Our expertise in ER stress, PERK, tau, AD, and TBI makes us uniquely suited to accomplish the pro- posed work. In addition, the unique resources available to my lab, such as small animal MR imaging, cohort biospecimens and clinical histories, and the UF Viral Production Core have strengthened the impact of our work and brought us closer to understanding the mechanisms of tau-mediated neurotoxic events stemming from the ER.

NIH Research Projects · FY 2025 · 2021-09

This proposal for the GenitoUrinary Development Molecular Anatomy Project (GUDMAP) will use the mouse model to investigate how differences between male and female external genitalia arise at the single cell level. Congenital malformations of the external genitalia (CAEG) are among the most prevalent human birth defects, affecting ~1:150 live male births. Hypospadias is a CAEG that is characterized by ectopic opening(s) of the urethra on the ventral side of the penis. In severe hypospadias, the urethral plate can open along the entire underside of the penis, giving it a clitoris-like appearance. This condition is termed ambiguous genitalia. Genetic causes of hypospadias have been elusive. The global prevalence has led to increased scrutiny of environmental factors, particularly endocrine disrupting chemicals (EDCs) that can induce genital anomalies in animal models. Sexual differentiation of the embryonic genital tubercle (GT) into a penis or a clitoris is regulated by gonadal androgens and estrogen. The central role of sex hormones in GT development creates sensitivity to EDCs; however, the mechanisms that mediate EDC effects on the GT are unknown, particularly at a cellular level. Do EDCs that cause hypospadias act on all cells in the GT, or do they target specific cell types? If subpopulation(s) of hormonally-responsive cells control urethragenesis, then buffering those cells against EDCs could be a method for prevention of hypospadias (analogous to folic acid’s ability to prevent neural tube defects). For genetic hypospadias, identifying cell-specific gene functions can distinguish direct regulators of urethragenesis from genes with indirect effects (e.g. sensitivity to EDCs). Understanding cell type diversity in developing external genitalia is essential for identifying the causes of CAEG and for developing preventative strategies. A major obstacle to progress in prevention and treatment of CAEG is the lack of knowledge of the specific cell types in external genital tissues. This project aims to fill these critical knowledge gaps by using single cell RNA-sequencing combined with novel imaging modalities to map cell type diversity in the developing external genitalia of normal male and female mice.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Brief motivational interventions (BMIs) to reduce risky drinking in college students are well established, but the needs of emerging adult (EA) risky drinkers who live in disadvantaged communities and are not fulltime college students have been neglected. They tend to have more constrained access to rewarding opportunities, adult roles, and activities that present pro-social alternatives to heavy drinking. When coupled with the foreshortened time horizons typical of many EAs, this suggests the need for interventions that not only enhance motivation to reduce drinking, but guide EAs to engage in alternatives to heavy drinking and orient their behavior toward longer-term positive goals. Guided by behavioral economics (BE), the proposed study will disseminate and evaluate a brief motivational BE intervention that combines BMI elements with the Substance Free Activity Session shown to reduce drinking by increasing future orientation and engagement in pro-social alternatives. The intervention will be delivered using a digital platform appropriate for EAs whose social networks operate through such communications. Because peers influence substance use, a peer-driven sampling method (Respondent Driven Sampling [RDS]) will be used to recruit 500 community-dwelling EAs ages 18-28 for a cluster randomized controlled trial that compares the intervention with an educational/assessment control condition. Drinking practices and problems, BE outcome predictors, and social networks will be assessed at baseline and at 1, 6, and 12-month follow-ups. Intervention efficacy and behavior change mechanisms will be examined. Reduced alcohol demand and delay discounting and favorable post-intervention shifts in future orientation, substance-free vs. substance-involved activities, and use of protective behavioral strategies to reduce drinking-related harms are predicted to mediate intervention effects. Social network analysis will assess whether the intervention attenuates network promotion of individual drinking. The study will be the first to test a web-based alcohol reduction intervention focused on BE principles and to use digital RDS to reach community- dwelling EAs for intervention. The study will translate and test BE mediators and moderators of change, and the digital intervention has high potential for reach and scalability with under-served community risk groups.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY: TRP channel regulation of feeding behavior in Drosophila Feeding is one of the most fundamental of all animal behaviors. In mammals, diverse signals, including chemosensory responses to food properties and mechanical stimulation of the gut, regulate satiation and feeding behavior. These mechanisms, or analogous systems, are well-conserved in invertebrates. Although many neuropeptides and neurotransmitters have been identified that signal satiation or satiety, much less is known about how gut stretch and other mechanosensory forces are signaled to regulate feeding behavior. Due to the remarkable conservation in their basic physiological and neurological properties, studies in Drosophila have revolutionized our broad understanding of animal behavior. Despite the successful use of flies in investigating paradigms such as sleep, circadian rhythm, and memory, only recently have tools for measuring fly food intake facilitated the study of feeding behavior. This project will use Drosophila melanogaster to investigate the regulation of meal intake. New methods allow undisturbed real-time measurements of food consumption in freely behaving adult flies that resolve with unrivalled accuracy the effects of diet and hunger on meal size. An experimental framework is established for defining fly meals and to show their basic regulation by manipulation of the diet, internal hunger state, and circadian rhythms. Using the powerful genetic tools available in Drosophila, preliminary results reveal the involvement of transient receptor potential (TRP) channels in the regulation of meal intake. TRP channels define a large family of sensory receptors, and much is still unknown about how they function as chemo-, mechano-, and other types of receptors. The proposed studies will dissect the role of TRP channel signaling in satiation and meal size control, taking advantage of the tools available in the simplified Drosophila model. Given the increasing importance of meal intake and patterning on physiology and health—regardless of total caloric intake—the proposed studies have the potential to reveal key insights on the role of TRP channel signaling on regulating prandial behavior, physiology, and metabolism. The proposed studies may also eventually inform the design of pesticides targeting TRP channels or feeding behavior in other insects, including agricultural pests and disease vectors such as mosquitos.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY DNA damage cannot be prevented because elements in a normal cellular environment including water and oxygen contribute to damage. Therefore, DNA repair pathways are essential for maintaining genomic stability and preventing diseases associated with DNA damage-induced mutagenesis. Even with functional DNA repair pathways, some DNA damage will inevitably escape repair and block DNA replication when encountered by the replisome. Cells have specialized pathways to bypass or repair DNA damage that blocks replication to allow replication to proceed. DNA helicases are among the enzymes essential for DNA repair, and these enzymes act both during and outside of DNA replication. The Rad3/XPD family of iron-sulfur (Fe-S)-containing DNA helicases plays an important role in DNA repair and maintaining genome stability. There are four family members in humans, XPD, FANCJ (a.k.a. BRIP1), DDX11 (a.k.a. ChlR1), and RTEL1, that are crucially important for human health as evidenced by diseases associated with mutations in each of the genes. Genetic disorders linked to mutations in Rad3/XPD family helicases are typically associated with genome instability, a predisposition to cancer, and a number of other pathologies. This proposal will define biochemical and molecular mechanisms for a newly discovered member of the Rad3/XPD helicase family in Escherichia coli, YoaA, that plays a role in repairing DNA damage during DNA replication. Our collaborators in the Lovett laboratory use 3’-azido- 3’deoxythymidine (AZT) as a reagent to block DNA replication in E. coli, and they identified two genes, yoaA and holC, that work together to give cells tolerance to AZT. Protein sequence predicts, and our preliminary results confirm, that the first gene, yoaA, encodes an Fe-S helicase. The second gene, holC, encodes the c subunit of DNA polymerase III holoenzyme (pol III HE) implicating the E. coli replicase in repair of AZT lesions. However, our preliminary results have uncovered a novel function for c as a subunit of the YoaA helicase, and we propose that c functions with the YoaA helicase rather than pol III HE in a pathway that repairs AZT lesions. The main premise of this proposal is that YoaA and c constitute a DNA helicase that is involved in the repair of damaged 3’ ends at stalled replication forks. Our aims are to: 1) characterize the YoaA•c protein, 2) characterize the helicase and substrate preferences for YoaA•c, and 3) define functional interactions between YoaA and c in vitro and develop a key reagent to investigate these functional interactions in vivo. This proposal will provide the first biochemical characterization of the YoaA•c helicase, a member of the Rad3/XPD family of helicases which play critical roles in human health by maintaining genome stability.

NIH Research Projects · FY 2025 · 2021-09

Abstract: The number of persons living with HIV (PLWH) continues to increase in the United States. Alcohol consumption is a significant barrier to both achieving the goal of ending the HIV epidemic and preventing comorbidities among PLWH, as it contributes to both HIV transmission and HIV-related complications. Recent advances in data capture systems such as mHealth devices, medical imaging, and high-throughput biotechnologies make large/complex research and clinical datasets available, including survey data, multi-omics data, electronic medical records, and/or other sources of reliable information related to engagement in care. This offers tremendous potential of applying “big” data to extract knowledge and insights regarding fundamental physiology, understand the mechanisms by which the pathogenic effects of biotic and abiotic factors are realized, and identify potential intervention targets. We propose to integrate the disparate data sources maintained by our partners and then utilize the big data to address research questions in treating HIV and alcohol-related morbidity and mortality. Specifically, we will pursue the following three aims: 1) Integrate the disparate data sources through standardization, harmonization, and merging; 2) Develop a web-based data sharing platform including virtual data sharing communities, data privacy protection, streamlined data approval and access, and tracking of ongoing research activities; 3) Provide statistical support to junior investigators to use the data repository for exploratory data analysis and proposal development. The proposed study will tap into disparate data sources, unleash the potential of data and information, accelerate knowledge discovery, advance data-powered health, and transform discovery to improve health outcomes for PLWH.

NIH Research Projects · FY 2025 · 2021-09

Abstract A reservoir of latently infected cells persists in various anatomical sites in people living with HIV (PLWH), despite effective virological control by antiretroviral therapy (ART). The majority of virally suppressed individuals experience rapid viral rebound upon ART interruption, providing a strong rationale for the development of cure strategies. Even in an ART-suppressed HIV infection, chronic inflammation and immune activation are observed, along with limited CD4+T cell reconstitution, mucosal immune dysfunction, co-morbidities, and accelerated ageing. Low-grade persistent transcription and trickling production of viral proteins from the pool of integrated proviruses are believed to be partly responsible for these conditions. HIV eradication strategies such as shock- and-kill have not been successful so far, and the pursuit of a functional cure or HIV remission has been thought as an alternative worth exploring. A functional cure entails long-term, durable control of viral expression in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. Our group has been at the forefront of developing one such strategy, labeled the block- and-lock approach. The premise of this approach is that transcriptional inhibitors can mediate epigenetic silencing of proviral expression, locking the virus in a profound state of latency from which reactivation is very unlikely to occur upon ART discontinuation. We have demonstrated this principle using the small molecule didehydro-Cortistatin A (dCA) inhibitor of Tat, the key regulator of HIV transcriptional amplification. In in vitro and in humanized mouse models of HIV latency, dCA inhibition of HIV transcription over time drives the viral promoter into deep transcriptional inhibition, limiting viral reactivation upon treatment interruption or with latency reactivating agents (LRAs)1–8. We believe that HIV transcriptional inhibitors, in general, have the potential to transform the way we treat HIV- 1 infections. Here we propose to investigate the potential of adding the transcriptional inhibitor dCA to an ART regimen in the rhesus macaque (RhM) model of SHIV infection. Not only is dCA a new molecule that inhibits the activity of a viral target not yet clinically explored, but it also opens the possibility for exploration of novel approaches to fight HIV. Here we propose to: 1) determine the safety and pharmacokinetics of dCA in ART- treated RhMs; 2) understand the relationship between dCA treatment and reduction in viral RNA in tissues, with the time to viral rebound upon treatment interruption; and 3) study the impact of dCA as front-line therapy on the size of the established viral reservoir.

NIH Research Projects · FY 2025 · 2021-09

Recent evidence put forth by our group and others suggests type 1 diabetes (T1D) pathogenesis involves a combination of immune, islet, and acinar pancreas defects. In addition to autoimmunity and β-cell death, it has become clear that T1D is characterized by a whole-organ pathology with reduced pancreas size, reduced exocrine enzyme levels in serum, and altered α- and β-cell function, including impaired insulin processing, even in the islet autoantibody positive (AAb+) pre-T1D condition. Hence, there is a need to understand each of these facets in concert, linking cellular phenotype and function, together with studies of the human pancreas tissue microenvironment, throughout T1D progression. We hypothesize that alterations to β-cell status and its surrounding environment are key determinants of impaired β-cell function, exocrine function, and infiltration (insulitis). We propose to assay islet and acinar tissue function using our novel pancreas slice culture platform (Aim 1a) to test (pro)hormone (proinsulin, insulin, glucagon) and enzyme (lipase, trypsinogen) secretion from T1D, AAb+, and control organ donor pancreata in response to established endocrine (glucose, arginine, KCl) and exocrine stimuli (cholecystokinin, carbachol). We will correlate these functional data with molecular features via scRNAseq (single cell RNA sequencing) with antibody-based CITEseq (Cellular Indexing of Transcriptomes and Epitopes) and scATACseq (single cell assay for transposase-accessible chromatin sequencing); this, for the purpose of cell identification together with transcriptomic and epigenomic analyses (Aims 1b). Pancreas slices will also be subjected to these same stimulatory conditions for live cellular imaging of Ca2+ signalling activity within islet and acinar tissue areas in real time (Aim 2a), then fixed and analyzed by imaging mass cytometry (IMC). We will assess in situ expression of 120 immune and pancreas cell markers with cellular resolution (Aim 2b), followed by spatial and temporal analysis of IMC data to determine how islet, immune and acinar cell phenotypes correlate with tissue and cellular function, using our histoCAT (histology topography cytometry analysis toolbox). This will enable computational analysis with 3D reconstruction from serial sections (Aim 2c). Finally, in Aim 3, human donor pancreas slices will be subjected to diabetogenic stimuli (inflammatory cytokines, glucotoxicity) and interventions targeting β-cell stress [imatinib (tyrosine kinase inhibitor), MSL-7 (autophagy enhancer), exenatide (GLP-1 receptor agonist)], and similarly evaluated by single cell and IMC profiling. With over 14 years of experience in procurement of transplant-quality human pancreata through the Network for Pancreatic Organ donors with Diabetes (nPOD), we are uniquely poised to perform the proposed studies. We expect to identify altered molecular pathways and tissue features linking β-cell, whole-islet, and acinar cell phenotypes with cellular function in AAb+ and T1D pancreata. We anticipate these same defects will arise in slices subjected to diabetogenic stimuli, providing a platform to test known and novel candidates for targeted intervention to reduce β-cell stress, restore islet and acinar cell function, as well as prevent disease progression.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Insufficient knowledge and throughput to interpret pathogenicity of genetic variants identified by next generation sequencing (NGS) is a major bottleneck for genomic medicine implementation. The American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP) guidelines identify high-confidence pathogenic and likely pathogenic variants but are limited in scalability. Many variants are classified as variants of uncertain significance by the ACMG/AMP guidelines without an indication of which of these variants are more or less likely to be pathogenic, leading to inappropriate medical treatment. Hence, I propose to develop standardized quantitative approaches to improve our ability to interpret genomic variations accurately at high-throughput. In-silico tools are commonly used to assign variant pathogenicity based on conservation, but their predictive accuracy is limited. The current methods have not been calibrated across genes, and the same pathogenicity score does not infer the same likelihood of pathogenicity across different genes. In this proposal, 1) I aim to recalibrate the pathogenicity scores incorporating gene-specific features making the pathogenicity scores more comparable across genes, and improve the accuracy of pathogenicity predictions using advanced deep neural network models and functional data from saturation mutagenesis studies. 2) I aim to quantify the ACMG/AMP variant classification and provide probability of variant pathogenicity for clinically relevant genes using advanced supervised learning and leveraging a large case- control cohort. The improved computational predictions (Aim 1) will refine variant prioritization for downstream analyses and strengthen the computational evidence used in the ACMG/AMP guidelines. The estimated probability of variant pathogenicity based on ACMG/AMP guideline (Aim 2) will improve communication between laboratories, health care providers and patients about genetic test results.

NIH Research Projects · FY 2025 · 2021-09

TITLE: Preclinical Assays of Hippocampal-Prefrontal Cortical Circuit Engagement for Application in Therapeutic Development FOA type: PAR-19-289: Abstract: The high failure rate of translating discovery science to positive clinical outcomes in the treatment of psychiatric diseases demonstrates the necessity of improving the efficiency and rigor of the therapeutic development pipeline. To this end, the critical importance of advancing the discovery of in vivo physiological and behavioral measures of the engagement of specific circuits for normal cognitive function has been acknowledged across funding initiatives. The hippocampus (HPC)-prefrontal cortical (PFC) circuit is critical for affective processing as well as higher cognitive functions and vulnerable in a number of mental health disorders. Although disrupted functional connectivity in the HPC-PFC circuit is a common feature of anxiety, bipolar disorder, schizophrenia, and autism, how local cellular interactions within this circuit manifest as large-scale temporal coordination to support higher cognitive functions remains unknown. Addressing this fundamental gap in our knowledge will establish a foundation for using circuit-based models for therapeutic target discovery and screening tools of novel drug efficacy. The long-term goal of this proposal, in line with the Funding Opportunity Announcement (PAR-19-289), is to enhance the therapeutic development pipeline for mental illness treatment by optimizing, evaluating, and mechanistically testing neurophysiological and behavioral measures of circuit engagement. The primary objective of this proposal, which is the first step towards achieving our goal, is to relate behavioral performance on the rodent analog on the Paired Associates Learning task (PAL), part of human Cambridge Neuropsychological Test Automated Batteries [CANTAB] assessment, and surface EEG recordings to invasive neurophysiological measures of neural coordination in the HPC-PFC circuit. Through an innovative series of experiments that integrate in vivo neurophysiological local field potential (LFP) recordings, circuit manipulation, surface EEG, and behavior, we will optimize, evaluate and mechanistically test novel noninvasive biomarkers of HPC-PFC circuit engagement by pursuing the following specific aims: 1) Optimize behavioral and non-invasive EEG biomarkers for inferring HPC-PFC circuit engagement and temporal coordination, 2) Evaluation of behavioral and non-invasive EEG biomarkers for determining HPC-PFC circuit engagement through pharmacological manipulation, and 3) Mechanistically test HPC-PFC projections as a driver of surface EEG organization. The proposed research is innovative because it integrates a clinically relevant behavioral task, designed to be analogous to human cognitive assessments, with surface EEG measures that translate across mammals. This will enable the optimization, evaluation, and testing of novel and translatable measures of HPC-PFC circuit engagement in the context of higher cognition and global neural organization. The significance of this contribution will be to provide novel diagnostic tools that can be used to enhance the therapeutic development pipeline for treating mental illness.

NIH Research Projects · FY 2025 · 2021-09

As persons living with HIV (PLWH) live longer, approximately 50% will experience HIV-related cognitive dysfunction, which may affect daily activities, contribute to morbidity and mortality, and increase the likelihood of HIV transmission. Alcohol consumption among PLWH may further exacerbate long-term cognitive dysfunction, with the presumed mechanism involving the gut microbiome, microbial translocation, systemic inflammation, and ultimately neuroinflammation. However, there are many gaps in our understanding regarding the specific pathophysiological mechanisms, and a need to offer interventions that are effective and acceptable in helping PLWH to reduce drinking or to protect them against alcohol-related harm. The overarching goal of this P01 is to identify and ultimately implement new/improved, targeted interventions that will improve outcomes related to cognitive and brain dysfunction in persons with HIV who drink alcohol. The proposed P01 activity will extend our current line of research that forms the core of the Southern HIV & Alcohol Research Consortium (SHARC). The specific aims of this P01 are to: 1) improve our understanding of the specific mechanisms that connect the gut microbiome to cognitive and brain health outcomes in persons with HIV; 2) evaluate interventions that are intended to reduce the impact of alcohol on brain and cognitive health in persons with HIV; and 3) connect and extend the research activity from this P01 with the training programs and community engagement activity in the SHARC. Our P01 will utilize two cores that provide infrastructure to two Research Components (RC1, RC2). The two RC will together enroll 200 PLWH with at-risk drinking into clinical trials that share common timepoints and outcome assessments. RC1 will compare two strategies to extend contingency management to 60 days, using breathalyzers and wrist-worn biosensors to monitor drinking. RC2 uses a hybrid trial design to evaluate two biomedical interventions targeting the gut-brain axis. One intervention is a wearable, transcutaneous vagus nerve stimulator that is hypothesized to stimulate the autonomic nervous system, resulting in decreased inflammation and improved cognition. The other intervention is a probiotic supplement intended to improve the gut microbiome in persons with HIV and alcohol consumption. All participants in RC2, and a subset of those in RC1 will have neuroimaging at two timepoints. The Data Science Core will provide data management and analytical support, and will analyze existing data and the data collected from this P01 using a machine learning and AI approach to identify factors associated with intervention success or failure. The Administrative Core will provide scientific leadership, clinical research and recruitment infrastructure, and connection to the outstanding training programs, development opportunities, and community engagement provided by the SHARC. Our community engagement with diverse populations, and collection of acceptability data from clinical trial participants, will facilitate our readiness to scale up the most promising interventions and move towards implementation in the next phase of our research.

NIH Research Projects · FY 2025 · 2021-09

Summary Compelling data support a contemporary version of the amyloid cascade hypothesis (ACH) as a valid framework both for understanding AD pathogenesis and the development of disease modifying therapeutics. However, key aspects of the ACH are not well understood. One such aspect is the relationship between accumulation of aggregated Aβ and neurodegeneration. The mainstream concepts regarding this relationship are that aggregates of Aβ are directly neurotoxic and/or trigger a toxic glial response. However, numerous observations indicate that the link between Aβ accumulation and neurodegeneration may be more complex. As a working hypothesis and a non-exclusive mechanism to the direct Aβ aggregate “toxin” model, we propose that a large number of biologically active proteins that we will refer to as amyloid associated proteins (AAPs) accumulate in the brain as Aβ deposits. Thus, Aβ aggregate accumulation may not be sufficiently toxic to induce downstream neurodegeneration unless accompanied by AAP accumulation. Indeed, in this scenario accumulation of AAPs helps to trigger the neurodegenerative phase of AD, accounting for the long delay between onset of Aβ deposition and neurodegeneration in humans. The proposed studies will leverage extensive data from the AMP-AD initiative and other published studies that has used state of the art proteomics to identify a large number of candidate AAPs that are increased both in AD and mouse models of Aβ deposition. Many of these candidate AAPs have known or inferred cell-signaling functions. Further, for some candidate AAPs there is either previous data demonstrating that they are associated with AD or we have generated novel data showing accumulation in senile plaques. Finally, as shown by others for the AAPs, ApoE and clusterin, we find that expression of select AAPs (midkine, pleiotrophin) modulates amyloid deposition. Building off this preliminary data, we propose three aims that are designed to probe our global hypothesis. In Aim 1 we will evaluate the spatiotemporal accumulation of AAPs in AD and in mouse models of amyloid deposition. In Aim 2 we will use rAAV- mediated expression of the AAPs in APP mouse models to a) further evaluate the association with amyloid plaques, b) determine if expression alters amyloid deposition and influences other AD relevant pathologies independent of effects on Aβ. In Aim 3 we intend to explore the mechanisms by which the AAP associates with the plaque and how that association might alter the biological properties of the AAP.

NIH Research Projects · FY 2024 · 2021-09

Artificial intelligence holds the promise of transforming data-driven biomedical research and computational health informatics for more accurate diagnosis and better treatment at lower cost. In the meantime, modern digital and mobile technologies make it much easier to collect information from patients in large scale. While “big” medical data offers unprecedented opportunities of building deep-learning artificial neural network (ANN) models to advance the research of complex diseases such as Parkinson’s disease (PD), it also presents unique challenges to patient data privacy. The task of training and continuously refining ANN models with data from tens of thousands of patients, each with numerous attributes and images, is computation-intensive and time-consuming. Outsourcing such computation and its data to the cloud is a viable solution. However, the problem of performing the ANN learning operations in the cloud, without the risk of leaking any patient data from their distributed sources, remains open to date. This application proposes to develop novel data masking technologies based on randomized orthogonal transformation to enable AI-computation outsourcing and data sharing, with the following two specific aims: 1) Perform two experimental studies of training ANN models with data masking in the HiperGator cloud for PD prediction and Parkinsonism diagnosis; 2) establish the theoretical foundation on data privacy, inference accuracy, and training performance of the ANN models used in the experimental studies. The interdisciplinary project team combines the expertise from data privacy, biomedical informatics, machine learning, and cloud computing to develop data outsourcing and sharing technologies for AI-powered PD research. The proposed research will remove a major roadblock that restricts medical data accessibility and hinders cloud-based operations of deep-learning artificial neural networks for biomedical research. The outcome is expected to have a broader impact beyond PD research in advancing the theory and implementation of cloud-based medical studies with data privacy protection.

NIH Research Projects · FY 2024 · 2021-09

Abstract: Glioblastoma multiforme (GBM), a grade IV glioma, is the most malignant form of an astrocytoma and is the most common malignant brain tumor in adults. The cause of this primary and highly aggressive cancer is unclear. The current treatment for glioblastoma is limited to maximal safe surgical resection, followed by chemotherapy and radiation therapy. Unfortunately, virtually all patients will have tumor recurrence and die of this disease. While survival without treatment is approximately three months, survival following treatment is only 12 to 15 months. Less than 5% of people survive longer than five years. A cardinal metabolic characteristic of tumorigenesis is a metabolic shift in which glycolysis, even in the presence of adequate tissue oxygen, increases disproportionately relative to oxidative phosphorylation (OXPHOS), a phenomenon known as the Warburg effect. This glycolytic shift occurs in GBM and is mechanistically associated with post-translational inhibition of the mitochondrial pyruvate dehydrogenase complex (PDC), which normally catalyzes the rate-limiting step in the aerobic oxidation of glucose-derived pyruvate and lactate. PDC inhibition is due to transcriptional upregulation of one or more of four pyruvate dehydrogenase kinase isoforms (PDK 1-4) that inhibit PDC by reversible phosphorylation. Dichloroacetate (DCA), the prototypic PDK inhibitor, readily crosses the blood-brain barrier and represents an entirely new class of small molecule metabolic modulators that act in mitochondria to reset cellular homeostasis in various congenital and acquired metabolic disorders. Indeed, pharmacological inhibition of PDK in cancer cells by DCA restores PDC activity, reverses the Warburg effect and induces a caspase-mediated selective apoptosis of tumors. Extensive pre-clinical research and early clinical trials in patients with recurrent GBM and other brain tumors indicate that DCA may be a safe and uniquely effective metabolic therapy for GBM. DCA inhibits its own metabolism and its only clinically limiting toxicity is reversible peripheral neuropathy. To mitigate this adverse effect, we developed and validated a genotyping method for genetics-based dosing of DCA that dichotomizes subjects into fast and slow drug metabolizers, leading to safe, personalized DCA dosing.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Development of platinum resistance is one of the most important factors contributing to ovarian cancer recurrence and mortality. In our effort to decipher molecular mechanisms underlying platinum resistance, we performed a kinome-wide screening on platinum-resistant SK-OV3 ovarian cancer cell line and identified Src- Related Kinase Lacking C-Terminal Regulatory Tyrosine And N-Terminal Myristylation Sites (SRMS) as a top platinum resistance regulator. Further analysis of TCGA ovarian cancer dataset revealed that patients with high SRMS expression responded poorly to platinum-based therapy and had worse overall survival. Since knockdown of SRMS markedly sensitized p53-deficient ovarian cancer cell lines to platinum while only displayed minor effect on p53-competent cell lines, we reason that SRMS plays a critical role in platinum resistance of p53-deficient ovarian cancer. To glean the mechanistic insight into the role of SRMS in platinum resistance, we showed that SRMS prevents JNK activation, possibly by directly phosphorylating JNKs. JNK signaling pathway is well established as an essential mediator for apoptosis triggered by cytotoxic agents; our observation that SRMS is specifically involved in platinum resistance in p53-deficient cells suggests that 1) platinum induces apoptosis in p53-deficient ovarian cancer cells in a JNK signaling pathway-dependent manner because of the defect in p53 signaling pathway-mediated apoptosis; and 2) SRMS-led inhibition of JNK signaling alleviates platinum-induced apoptosis and thereby promotes platinum resistance in p53-deficient cells. p53 is uniformly deficient in high grade serous ovarian cancer (HGSOC). The discovery of SRMS’ prominent role in platinum resistance of p53- deficient cells indicates that SRMS can be an ideal therapeutic target against platinum resistance in HGSOC. In our “drug repurposing” screening, we found that PLX4720, a selective inhibitor of B-RafV600E, can potently inhibit SRMS activity. In this application, we propose 3 specific aims: 1) Characterize molecular mechanisms underlying SRMS-conferred platinum resistance; 2) Define platinum resistance-relevant events that are governed by SRMS-JNK signaling; and 3) Investigate the potential of targeting SRMS to augment efficacy of platinum therapy. The success of this application will uncover molecular mechanism underlying SRMS’ role in platinum resistance of ovarian cancer. Importantly, we will evaluate SRMS-targeted strategy to overcome platinum resistance in ovarian cancer.

NIH Research Projects · FY 2025 · 2021-09

Symptomatic intracranial atherosclerotic stenosis (sICAS) is a common disease associated with a very high risk of stroke. Although clopidogrel + aspirin and intensive risk factor management are considered standard care for sICAS, the 1-year rate of all stroke and vascular death in subjects presenting with a symptomatic infarct and 70- 99% sICAS was 27% with this therapy in the SAMMPRIS trial. Clearly, we need better treatment. Combining ticagrelor with aspirin may be more effective than clopidogrel + aspirin for sICAS because ticagrelor provides faster, greater and more consistent platelet inhibition than clopidogrel. Additionally, ticagrelor is a direct P2Y12 receptor antagonist and may be more effective than clopidogrel in patients who carry genetic single-nucleotide loss-of-function (LOF) polymorphisms for the CYP2C19 cytochrome P450 enzyme necessary to metabolize clopidogrel to its active form. The novel oral anticoagulants (NOAC) may also offer potential advantages in patients with sICAS. Atherosclerotic disease progression to an unstable state is characterized by increased platelet activation, elevated procoagulant activity and thrombin generation, which provides the mechanistic rationale for combining anticoagulation with an antiplatelet agent in patients with atherosclerosis. However, combining full dose anticoagulation with an antiplatelet agent increases the risk of major hemorrhage, including intracerebral hemorrhage (ICH). This has led to interest in combining a low dose NOAC with low dose aspirin in patients with atherosclerosis. We propose a seamless Phase II/III adaptive, prospective, double-blinded, 3-arm clinical trial at 115 sites that will randomize 1683 high-risk subjects with sICAS to 1 year treatment in one of three arms: 1) ticagrelor (180 mg loading dose, then 90mg twice daily), 2) low dose rivaroxaban (2.5mg twice daily), or 3) clopidogrel (600mg loading dose, then 75 mg daily). All subjects will also receive aspirin (81mg daily) and intensive risk factor management per the SAMMPRIS protocol. The 3-arm Phase II/III adaptive design increases the efficiency with which we can evaluate two new potential therapies for sICAS, using a shared control group and a shared trial infrastructure. The Phase II Primary Aim is to identify an excess of ICH or non-ICH major hemorrhage in the rivaroxaban or ticagrelor arms that could lead to an early termination of one or both of those arms. The Phase III Primary Aim is to determine if the experimental arm(s) (rivaroxaban or ticagrelor or both) that progress from Phase II to Phase III are superior to the clopidogrel arm for lowering the 1-year rate of the primary endpoint (ischemic stroke, ICH, or vascular death) in subjects with 70-99% sICAS. The Exploratory Aim is to estimate the impact of CYP2C19 LOF carrier status on any benefit that the ticagrelor or low dose rivaroxaban arms may have in lowering the primary endpoint compared with the clopidogrel arm. This innovative trial will evaluate two new antithrombotic approaches to maximize the chance of establishing more effective therapy for sICAS, one of the most common and high-risk cerebrovascular diseases worldwide.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Type 1 Diabetes (T1D) is an autoimmune disease caused by aberrant T-cell mediated targeted destruction of insulin-producing beta cells in the pancreas, resulting in loss of blood glucose regulation, with increased long- term risks of vascular and neuropathic comorbidities. Despite the fact that T1D is one of the most studied organ- specific autoimmune diseases, the various strategies aimed at intervention, prevention, or reversal of this disease have failed to succeed due to incomplete knowledge about the precise mechanisms of their action, as only peripheral assessments of systemic impacts (e.g., circulating cytokine changes, C-peptide levels) are feasible. This lack of mechanistic understanding of these interventions, as well as substantial time and cost of clinical trials, is a profound obstacle in improving therapeutic outcomes. To address these significant knowledge gaps, there is a substantial clinical need to develop human-based ex vivo systems capable of intimately studying the interplay of islets and immune cells, as well as the contribution of environmental factors on immune cell activation, homing, and cytotoxicity. The primary hypothesis of this proposal is that the development of an islet- immune platform has the potential to provide unique insight into T1D, with investigation of activation pathways and screening of interventional approaches. Thus, the objective of this proposal is to engineer, validate, and utilize a unique in vitro 3-D platform for the interrogation of human T1D immunopathogenesis by converging innovative cells with biomaterials, in situ imaging, and microphysiological systems (MPS). Aim 1 will seek to establish and validate this 3D biomaterial-based co-culture platform. To validate the system, a tiered approach, building from single antigen murine model cells to human T1D-antigen cells, will be employed. Once validated, Aim 2 will translate this platform to study human-centric T1D-relevant pathways and interventions. Finally, Aim 3 will seek to integrate spatial and fluidic features by translating the 3D material to an established microphysiological system (MPS) platform, which will permit the study of T cell migration from a fluidic microenvironment to the beta cell niche. Results from this proposal should provide a validated and enabling tool for the study of human T1D-relevant pathophysiology, interventions, and therapeutics. While the proposed field of application for this platform is T1D, other autoimmune diseases can benefit from this engineered benchtop platofrm, as they share homologous hallmarks of immune cell dysregulation.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT The U.S. healthcare system cares for increasing numbers of older adults, including those with Alzheimer’s disease and related dementias, who require surgery with anesthesia. Regional social inequities, as measured by spatial indices such as area deprivation and social vulnerability indices, independently contribute to differences in perioperative and neurodegenerative outcomes. Data also support a significant association between neighborhood-level area deprivation indices with Alzheimer’s disease neuropathology. Despite emerging evidence linking social determinants of health with disparities in postoperative outcomes in older adults, investigations in these areas increasingly require expertise with disparate subject matters, including clinical perioperative practice, geriatrics, neuropsychology, social determinants of health, geospatial analyses, and even artificial intelligence. The K07 applicant is a National Institutes of Health (NIH)-funded independent investigator and fellowship-trained anesthesiologist with expertise in regional anesthesia and orthopedic surgery, as well as research experience in machine learning, geospatial disparities, and perioperative pain and cognition. The purpose of this proposal is to promote institutional and community awareness of how social vulnerabilities impact perioperative outcomes in older adults, including those with Alzheimer’s disease and related dementias, and particularly those outcomes related to postoperative pain and cognition. The overall strategy of this proposal is to develop interdisciplinary research infrastructure along with transdisciplinary training programs to crosspollinate research teams. This structure will be organized via the Perioperative Cognitive Anesthesia Network-Social Vulnerability (PeCANSV) construct in three aims: Aim 1: Convene a PeCANSV multidisciplinary advisory team to establish three supplemental cores in 1) geospatial infrastructure; 2) social determinants of health; 3) artificial intelligence. Aim 2: Accelerate research discovery through interdisciplinary research pathways by formalizing nascent linkages between supplemental PeCANSV cores and UF resources and extant geospatial, environmental, cognitive, and clinical perioperative databases at UF Health and throughout Florida. Aim 3: Develop a transdisciplinary workforce of clinical and basic science researchers to lead interdisciplinary teams addressing the social vulnerability of older adult surgical patients. Milestones include regular analysis of strengths, opportunities, aspirations, and results, interdisciplinary proposal submissions, and evidence of trainee success in perioperative research in social disparities of older adults, including those with Alzheimer’s disease and related dementias.

NIH Research Projects · FY 2026 · 2021-09

Preemptive pharmacogenetic (PGx) testing may be particularly beneficial in medically underserved populations by reducing the number of appointments required to optimize drug therapy and increasing the effectiveness of less expensive off-patent medications – the type most often with pharmacogenetic guidelines available (PGx drugs). However, there is little data available to guide clinical implementation in these patient populations. Our long-term goal is to contribute toward the efficient implementation of PGx into clinical practice to improve the precision of medication prescribing. The overall objective for this application is to identify PGx drug usage patterns in medically underserved patients and assess the feasibility and effectiveness of preemptive PGx testing in this patient population. The central hypothesis is that medically underserved patients are prescribed more PGx drugs, and preemptive PGx testing is feasible as well as effective in improving patient medication satisfaction. The rationale for the proposed research is that identifying patient populations that can most benefit from PGx testing will facilitate clinical implementation that may reduce medication treatment disparities. We plan to test the central hypothesis and accomplish the overall objective of this application by pursuing three specific aims. The first aim is to identify clinical, demographic and socioeconomic factors associated with PGx drug prescribing patterns in a large, real-world patient population. We will accomplish this aim by comparing clinical, demographic, and socioeconomic data with prescription data from millions of patients across the State of Florida. The second aim is to develop a low-cost, genetic ancestrally comprehensive PGx testing panel designed to inform commonly used PGx drugs. We will design a low-cost, clinically validated panel that will include variants multiple genetic ancestral populations in the U.S. We plan to leverage extensive batching of tests and an already available genotyping platform that minimizes labor costs in order to achieve significant cost savings. The third aim is to determine the feasibility of low-cost preemptive PGx testing in a medically underserved population as well as its effect on patient medication satisfaction. We will accomplish this aim by completing a randomized open-label clinical trial comparing medically underserved patients receiving preemptive PGx testing to those receiving usual care. We will compare key implementation metrics and will also conduct semi-structured interviews in both patients and healthcare providers to assess PGx perceptions of feasibility and sustainability from stakeholders. The proposed research is significant because it should contribute valuable preliminary data toward both the real-world effectiveness of preemptive PGx testing as well as the feasibility of studying and implementing this technology in medically underserved patients – an area of PGx research where few data are available. The proposed research is innovative because this project will utilize additional demographic and socioeconomic data that, along with clinical data, should better identify patient populations most likely to benefit from PGx testing and allow focused implementation efforts in those populations. Ultimately, we expect to have developed valuable data identifying patients most likely to benefit from preemptive PGx testing, particularly in patients who are medically underserved. These results should have an important positive impact because they can inform further clinical implementation efforts of PGx as well as future large clinical trials of preemptive testing, ideally reducing healthcare disparities in the field of precision medicine.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Our long-term goal is to foster genetically-informed reproductive health knowledge and behaviors among young adults with sickle cell disease (SCD) or sickle cell trait (SCT) with a web-based, tailored, multimedia intervention (CHOICES). In follow-up to genetic counseling, CHOICES helps them engage in behaviors concordant with their parenting plan. Developed with the sickle cell community, CHOICES is designed to help young adults with SCD or SCT gain knowledge of genetic inheritance, specify their preconception reproductive health intentions (their parenting plan), and engage in reproductive health behaviors concordant with their parenting plan. We found high acceptability of both the e-Book (usual care control) and CHOICES interventions. We also found sustained (24 mo), significant effects on knowledge but not on at behavior, presumably because 49% of the participants were not at risk of having a child with SCD (e.g., partner had normal hemoglobin, Hgb A). Of the participants at risk of having a child with SCD (at-risk) and who expressed intention to get pregnant soon and have a child free of SCD, the CHOICES group’s (n=26), behaviors showed a 27% relative at-risk time reduction compared to the e-Book group (n=16). However, the difference was not statistically significant given the small, underpowered sample (n=42). Hence, we propose an adequately powered RCT with the CHOICES intervention and an e-Book control to compare their effects on genetic inheritance knowledge and at-risk reproductive health behaviors (immediate posttest and at 6, 12, 18, & 24 mo). Using proven strategies (referrals, social media, and communication technology) for recruiting and retaining the sample, we will use web-based data collection (SCKnowIQ) and intervention delivery strategies enhanced by nudges and tailored boosters. In a sample of 430 adult men and women, aged 18-35 yr with SCD or SCT, at-risk, and planning within 2 years to have a child free of SCD, the specific aims are to: Aim 1. Compare e-Book and CHOICES groups for effects on (a) knowledge (primary endpoint), and (b) at-risk behavior (secondary endpoint) measured with the SCKnowIQ over time (baseline, post-test, 6,12, 18,24 mo). We hypothesize that across all time points post baseline, there will be (a) higher knowledge scores and (b) lower percentage of time with at-risk behaviors (at-risk time) in the CHOICES than in the e-Book groups. Aim 2. Explore the relationship between demographic factors (e.g., sex, SC status), intervention acceptability, and the Aim1 endpoints (knowledge, behavior). We will conduct subgroup analyses to provide insight into the baseline knowledge and behavior as well as the intervention effects in different demographic or acceptability groups. Given the scalability and low cost of CHOICES, if proved to be effective, it can reach the affected population at low cost. As a genetic counseling follow-up model, CHOICES may translate into informed parental decisions and preparedness for the consequences of their preconception decisions. Considering the implications for SCD and other single-gene diseases globally, the potential impact is enormous.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Tick-borne rickettsial diseases (TBRDs) are ubiquitously present throughout the world and case fatality rates in disease clusters can range up to 100% despite the availability of effective treatment. Thus, there is a need to increase the “tool box” for TBRD control by supplementing existing strategies with promising novel approaches that focus on interrupting the Rickettsia transmission cycle in the tick vector. Our recent studies demonstrated that the non-pathogenic Candidatus Rickettsia andeanae is secreted in tick saliva during feeding, but at a lower level when compared to pathogenic Rickettsia parkeri, raising questions regarding the underlying mechanisms that mediate rickettsial pathogenicity. The likelihood that rickettsiae manipulate the arthropod host to enhance horizontal transmission has been recognized, yet the specific interactions between Rickettsia and ticks remains unknown. Unfortunately, significant knowledge gaps exist regarding the basic transmission biology of tick-borne Rickettsia and the specific interactions between Rickettsia and tick physiology that enable transmission, which represents a significant barrier to the field and has limited the development of novel approaches to control TBRDs. Thus, the premise of this proposal is that pathogenic Rickettsia, but not non-pathogenic strains of Rickettsia, alter regulation of tick-derived proteins to enhance salivary gland physiology and increase the secretory activity of the gland, which facilitates increased horizontal transmission. Salivary gland function is dependent on strict regulation of acini membrane physiology and thus, we hypothesize that Rickettsia alter mechanisms for ionic homeostasis to facilitate horizontal transmission. Correspondingly, we hypothesize that inhibition of ion flux will negate the rickettsial-mediated enhancement of salivary gland activity to prevent tick bloodmeal feeding and horizontal transmission of Rickettsia. In Specific Aim 1, we will employ a multidisciplinary approach to measure the influence pathogenic Rickettsia has to secretory activity and membrane physiology (e.g. membrane potential) of an R. parkeri infected tick salivary gland compared to non- pathogenic Rickettsia infected ticks. These data will delineate the mechanism by which Rickettsia influences tick salivary gland physiology to drive pathogenicity. In Specific Aim 2, we will test if dysregulation of K+ homeostasis across salivary gland epithelia will inhibit salivary gland function of rickettsemic ticks to alter blood feeding biology and reduce R. parkeri virulence in vertebrate disease models. Combined, the experiments outlined in his proposal will define unique aspects of rickettsial influence on tick physiology that enhance pathogenicity of Rickettsia, which will assist in resolving the epidemiology of SFG Rickettsia and reveal intervention points to reduce the health burden of TBRDs.