University Of South Florida

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Staphylococcus aureus is a pervasive pathogen that causes invasive disease in humans. The type 7b secretion system (T7bSS) of S. aureus transports a specific set of factors across the bacterial envelope, which are required for S. aureus virulence and persistence in infected host tissues. Secretion of these proteins is dependent on the expression of the ess operon, which encodes T7bSS structural proteins that assemble into a transport complex within the bacterial envelope. The mechanisms of T7bSS expression and assembly are not well understood. Dr. Bobrovskyy's research established a purification protocol for the T7bSS complex and identified accessory gene regulator (Agr) and peptidoglycan hydrolase EssH as factors necessary to support T7b secretion. Thus, the overall objectives of this proposal are to reveal the composition, stoichiometry and assembly of the purified T7bSS complex (Aim 1), determine the mechanism whereby peptidoglycan hydrolase EssH supports T7b secretion (Aim 2), and elucidate T7bSS regulation by Agr (Aim 3). Together, these aims will test an overarching hypothesis that the ess locus of S. aureus is regulated by the Agr pathway, leading to the assembly of the T7bSS complex that spans the envelope and permits substrate translocation. In Aim 1, purification of the T7bSS complex coupled with single-particle electron cryomicroscopy (cryo-EM) and cross-linking mass spectrometry will be utilized to investigate the structural components that enable translocation of substrates across staphylococcal cell envelope. In Aim 2, a combination of genetic and biochemical approaches will be used to investigate the contribution of EssH to the assembly of T7bSS complex and substrate translocation across a thick peptidoglycan cell wall. In Aim 3, the role of the post-transcriptional regulator RNAIII, a component of the staphylococcal Agr pathway, and of other intermediate factors, will be examined for ess gene regulation. In addition, the proposed training and career development activities are intended to provide Dr. Bobrovskyy with the experience and tools that will allow him to successfully transition to independence in the field of bacterial physiology and pathogenesis. The collaborative and interdisciplinary research environment in the Department of Microbiology at the University of Chicago, and access to the state-of-the-art equipment at the core facilities, such as the Advanced Electron Microscopy Facility, are well suited for the candidate's proposal. The candidate's mentors Drs. Missiakas and Zhao, will assure the progress of the research and training objectives. Dr. Missiakas is an internationally recognized scientist in the field of staphylococcal protein secretion, with a strong history of mentoring trainees, many of whom went onto having careers in academia and industry. Dr. Zhao is a structural biologist who specializes in cryo-EM analysis of membrane protein complexes and will provide training and support to the candidate in this area. Dr. Bobrovskyy also assembled an advisory committee consisting of Drs. Phoebe Rice, Jim Slauch and Sam Light, who will assist the candidate in his research and training. Overall, this award will enable Dr. Bobrovskyy to attain his goals and propel his career towards independence.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Neural biomarkers are an important focus of the NIMH Research Domain Criteria (RDoC) initiative, and they are increasingly used in the context of genomic studies and clinical trials. The use of biomarkers with strong psychometric reliability increases the likelihood of finding replicable effects, improves the validity of their interpretation, and decreases the likelihood of missing real phenomena. Although fundamental psychometric principles have long been a prominent concern among studies that use self-report measures, these principles are underappreciated in studies of psychopathology that use biological measures. This lack of attention to reliability limits the more widespread application of biomarkers in psychopathology and likely contributes to replication problems. Generalizability theory is a multifaceted framework for identifying sources of measurement error, and this framework is uniquely suited to assessing the reliability of biological measures and to optimizing tasks for reliability. A critical need exists for tractable software to facilitate the application of generalizability theory to time-frequency electroencephalography (EEG), event-related potentials (ERPs), facial electromyography (EMG), and electrodermal activity (EDA). The objective of this project in response to PAR- 18-930 on measurement tool development for RDoC is to (i) develop an extensive treatment of generalizability theory for psychopathology researchers, (ii) develop accessible software to implement it, (iii) show how to apply these resources to optimize paradigms for individual-differences research, and (iv) disseminate the software with a user-friendly guide. This project will facilitate the routine evaluation of reliability through these specific aims: 1) Design and implement generalizability theory formulas for evaluating group- and subject-level reliability for paradigm optimization; 2) Develop software to implement these formulas with data from widely used psychophysiological software; 3) Apply results to optimize three commonly studied tasks; and 4) Develop online educational material on the application of these resources to novel paradigms and measures. This research project is innovative, because it represents a substantive departure from standard practice by shifting the focus to the reliability of data from individuals, rather than groups, to identify sources of measurement error and minimize their impact. This work promotes best practices in reporting psychometric properties of biological measures and is applicable to data from any task with trial-wise scores. The resulting open-source toolbox, the Psychophysiologist’s Reliability Analysis Toolbox (PsyRAT), can facilitate guidelines for optimizing paradigms, making decisions about individual-subject data, and grounding individual-differences questions (central to clinical research, especially for applications in precision medicine) in measures of reliability. The proposed process for guiding biomarker evaluation through high-quality psychometrics will pave the way for better selection of biomarkers and task development, ultimately improving the clinical utility of these biomarkers.

NIH Research Projects · FY 2026 · 2022-08

Despite ample evidence that adolescent alcohol abuse differs dramatically from adult alcohol dependence in terms of both drinking habits and treatment needs, few interventions address the unique circumstances of the typical teen. More than half of teenagers do not get enough sleep on a regular basis. This sleep disruption increases sensitivity to stress and drives alcohol consumption in response to both sleep problems and anxiety. 19% of 12- to 20-year-olds report binge drinking in the past month, and 30% drink alcohol on a regular basis. In addition to immediate risks such as academic difficulty, car accidents and even violence, almost half of these adolescents will struggle with alcohol dependence at some point in their lives. To reduce alcohol abuse, we need to improve sleep and reduce the stressors that lead adolescents to over-indulge in the first place. We have developed a novel protocol to generate a weekday-weekend sleep pattern in adolescent mice, in order to model circadian sleep disruption and study the ensuing effects on stress and alcohol intake. The proposed studies will examine how circadian desynchrony and sleep homeostasis impact the self-perpetuating cycle of sleep disruption, stress, and alcohol drinking in adolescence, the period of greatest vulnerability to the neurobiological changes underlying addiction, and the long-term effects of this cycle on drinking in adulthood. Next, we will test whether melatonin, which resets the internal clock and serves as a potent systemic cue for the switch from daylight to nighttime physiological patterns, can restore sleep and reduce both stress and alcohol intake in our models. In the first Aim, circadian phenotyping cages will be used to generate circadian desynchrony, then we will assess alcohol drinking and sleep patterns, stress reactivity, and alcohol seeking after punishment in adolescent male and female melatonin-proficient C57BL/6 mice, and we will correlate behavioral patterns with the rhythmic release of corticosterone and melatonin, as well as with the rhythmic expression of circadian genes and corticotropin releasing factor receptors. In a second group, we will follow the same adolescent protocol, allow mice to age, then test the same behavioral measures in adulthood. In the second Aim, we will use timed sleep restriction to awaken adolescent mice earlier than their natural wake-up time and follow the same assessments as in Aim 1. We propose that circadian sleep disruptions will increase alcohol intake by disrupting sleep, increasing stress activation, and increasing motivation to work for alcohol despite punishment. In Aim 3, we will further explore the effects of alcohol and circadian sleep disruption on stress by examining how these factors impact the activity of the molecular stress axis itself. This will provide an essential foundation of knowledge about the interactions between the circadian sleep system and the stress axis in adolescent alcohol abuse, the long-term effects of these interactions, and the potential for melatonin to both reduce alcohol intake in adolescents and to reduce the risk of developing alcohol use disorders later in adulthood.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Advances in computational and experimental protein engineering have ushered in a new era of biomolecule development, providing new functional binding proteins, improved enzymes, and rationally designed synthetic receptors. Despite such progress, established techniques lack the ability to develop and study synthetic receptors and inhibitory molecules in high throughput, instead relying on rational or function-agnostic engineering approaches followed by low-throughput characterization for the desired activity. This deficiency in approach results in 1) the development of suboptimal candidate proteins and 2) a costly development process. My long-term goals are to 1) establish new platforms that incorporate protein function as a selective pressure to engineer new molecules and 2) utilize these new platforms to understand the roles of natural and synthetic proteins in cell signaling responses in both normal and disease pathologies. I hypothesize that the incorporation of protein function as a selective pressure in protein engineering campaigns will result in the efficient development of new classes of functional proteins capable of answering key biological questions. The goals during this proposal period are to establish high-throughput screening platforms for inhibitor engineering and for synthetic receptor engineering, as well as develop combined computational and experimental protein engineering pipelines to aid the study of important proteins. Continued work in the described areas has immense potential to aid the study of basic and synthetic biology through 1) greatly expanding the availability of molecules to empower studies of the importance of individual molecules in cell signaling responses, 2) providing a new toolkit for understanding synthetic receptor function, and 3) providing modular platforms to aid discovery of functional engineered molecules. We will pursue three primary directions: Direction 1: Establish a tethered inhibitor engineering platform with yeast surface display. Current directed evolution approaches often lack protein function as a selective pressure, resulting in majority development of passive binding proteins. We will incorporate the concept of tethering from the small molecule screening community to drive yeast-displayed protein selection toward active inhibition. Direction 2. Establish combined computational and experimental protein engineering pipelines. Current protein engineering approaches typically rely on widespread mutagenesis or in-depth computational/rational design to develop functional proteins. We will incorporate accessible computational approaches for protein mutant library design and investigate these approaches for basic mutational studies. Direction 3. Establish a high-throughput platform for studying synthetic receptors. Synthetic receptors have made measurable impacts in both basic and clinical science, but no effective platform exists to study their development in high throughput. We will establish a high-throughput screening platform with proliferation from receptor functions as a selective pressure for engineering new synthetic receptors.

NIH Research Projects · FY 2026 · 2022-07

Project Summary/Abstract Of over 80,000 adolescents and young adults (AYA) diagnosed with cancer in the United States each year, 85% live for at least five years after their cancer diagnosis. Adolescence and early young adulthood is a critical period for frontal neurodevelopment, and cancer treatments potentially disrupt this neurodevelopment, leading to cognitive deficits known as cancer-related cognitive impairment (CRCI). An estimated 53% of these long-term early young adult (YA) survivors experience CRCI that interferes with their work and educational goals. However, sparse research using objective neuropsychological assessment does not detect CRCI as reported by YA survivors. Therefore, the goal of this K99/R00 application is to take the first step in an innovative program of research to characterize CRCI among this vulnerable survivorship population using ecologically valid assessments. Career Development Plan: The overall training objective is to provide Dr. Tometich with additional training and mentorship to become a highly qualified independent investigator with expertise in CRCI in the understudied population of AYA cancer survivors. Dr. Tometich’s training goals are to: 1) enhance her knowledge of cancer and cognition in AYAs, 2) acquire skills in cognitive and behavioral assessment methodologies, 3) develop proficiency in real-time longitudinal data analysis, and 4) continue professional development. During the K99 phase, Dr. Tometich will work under the primary mentorship of Dr. Heather Jim at Moffitt Cancer Center (an NCI-designated comprehensive cancer center) and co-mentorship of Dr. Brent Small. Experts in CRCI among AYA survivors are lacking nationally; therefore, a mentorship and advisory team will provide complementary expertise to address each aspect of the training and research content (additional advisors are Drs. Andrew Galligan, Martin Sliwinski, and Dinorah Martinez Tyson). Research Plan: The goal of this study is to evaluate long-term CRCI and potentially modifiable risk factors in YA survivors (i.e., age 18-30) using ecologically valid assessments. An existing cognitive ecological momentary assessment (EMA) has been developed and used by our research team in middle-aged breast cancer survivors, but it has not yet been applied in YA survivors. Furthermore, actigraphy can objectively measure two modifiable risk factors for CRCI (physical activity and sleep). In the K99 phase (i.e., Aims 1 and 2), we will use an iterative mixed-methods approach to tailor an EMA of CRCI and situational, behavioral, and contextual risk factors (SBCF) to the experience of YAs based on qualitative interviews with 20 YA survivors who report CRCI. We will then pilot the EMA and actigraphy in 25 YA survivors and make revisions as needed. In the R00 phase (i.e., Aim 3), we will recruit 150 YA survivors and 150 community controls matched on gender, age, and education for a cross-sectional study to evaluate CRCI and SBCF. Impact: The combined training and research plan will position Dr. Tometich to transition to independence as one of the few cancer control investigators specializing in CRCI in the vulnerable and unique population of AYA survivors.

NIH Research Projects · FY 2025 · 2022-07

This R35 MIRA grant application addresses a fundamental gap in understanding of the mechanisms that underlie trauma-induced microvascular leakage, a hallmark of the systemic inflammatory response. The long-term goal is to identify novel targets that can be used to ameliorate microvascular leakage in the context of traumatic injury, in order to improve outcomes for trauma patients. To achieve this goal, the current knowledge of the cellular and molecular signals that control microvascular permeability must be significantly expanded, including signals that promote hyperpermeability and those that promote resolution toward normal barrier function. Also, very little is known about how alcohol intoxication, which often accompanies traumatic injury, worsens microvascular leakage leading to poorer outcomes for trauma patients. Until these gaps in knowledge are filled, physicians will not be able to shift beyond current therapeutic paradigms to the next level of care required to save many patients that worsen over time after trauma, developing sepsis and multiple organ failure. To significantly expand the current knowledge base of how microvascular hyperpermeability develops and is resolved, the proposed research capitalizes on emerging approaches that have become more widely available. These include RNA-Seq, proteomics, metabolomics, and lipidomics, which provide unbiased analysis of changes in expression of genes and the molecular landscape. Applying these methods to experimental models of trauma or cells/tissues from trauma patients will identify novel molecules associated with trauma-induced microvascular hyperpermeability that will reveal answers to three key questions that must be addressed in order to advance new therapies: 1) Which endothelial signals activated by alcohol intoxication and hemorrhagic shock sustain increased microvascular leakage, and which terminate microvascular hyperpermeability? 2) Can sustained microvascular hyperpermeability be accurately predicted and monitored using plasma biomarkers of endothelial injury or leukocyte activation, to help guide therapeutic interventions? 3) How can fluid resuscitation be optimized to reduce microvascular hyperpermeability, improve blood-tissue exchange, and better prevent organ dysfunction? A multilevel approach will be used to answer these questions featuring an established, clinically relevant rodent model of combined alcohol intoxication and hemorrhagic shock/resuscitation, supported by cultured endothelial cell models that will increase the depth of understanding about how the microvascular endothelium responds to trauma/shock. This proposal also leverages the PI’s unique expertise with isolating intact venules for study, and to maximize translational impact will utilize a novel human isolated venule permeability model. Finding answers to these key questions is important, because having comprehensive knowledge of the signals that activate and terminate microvascular hyperpermeability, the biomarkers involved, or what key factors in plasma are endothelial barrier-protective, will permit logical development of new, personalized therapeutic strategies to extend and improve life.

NIH Research Projects · FY 2025 · 2022-07

Myocardial infarction (MI)-induced heart failure is the leading cause of morbidity and mortality in the United States. About one in four MI patients will progress to develop chronic heart failure, which has a 5-year mortality rate of 40%. It is highly urgent to improve long-term outcomes of MI patients. Lymphopenia, a reduction in peripheral blood lymphocyte count (primarily due to T-cell loss), has consistently been shown to correlate with worse cardiac function and poor outcome in MI patients and is an independent marker to predict the prognosis. Unfortunately, how T lymphopenia occurs following MI and whether targeting T lymphopenia has therapeutic potential are largely unknown. Using the mouse MI-induced ischemic heart failure model, our preliminary data showed that MI-induced T lymphopenia may involve blood T-cell redistribution to the bone marrow and T cell development impairment. CD4+ T-cell activation is known to improve wound healing post-MI. Thus, persistent CD4+ T lymphopenia may reduce protective CD4+ T-cell response and compromise myocardial repair after MI. The goals of this proposal are: 1) to elucidate the underlying mechanisms that cause T lymphopenia following MI; and 2) to investigate whether inhibiting CD4+ T lymphopenia can serve as a therapeutic strategy. We hypothesize that MI induces T lymphopenia by both stimulating T-cell trafficking from blood to the bone marrow and impairing T lymphopoiesis; inhibiting CD4+ T lymphopenia improves post-MI cardiac repair. Three specific aims will address this novel hypothesis in a mouse ischemic heart failure model that combines multidisciplinary approaches. Specific Aim 1 will examine the mechanisms of blood T-cell trafficking to the bone marrow and alterations of distinct T-cell phenotypes and functions after MI. Specific Aim 2 will determine the mechanisms by which MI impairs T lymphopoiesis. Specific Aim 3 will examine the hypothesis that inhibiting CD4+ T lymphopenia would improve post-MI cardiac repair. Accomplishment of this proposal will provide new insights into T lymphopenia mechanisms in ischemic heart failure and may offer potential intervention strategies to improve the prognosis of heart failure patients.

NIH Research Projects · FY 2025 · 2022-07

Congenital lymphedema is caused by inherited gene mutations that impair the functioning of the lymphatic vasculature and lead to swelling of the limbs, disfigurement, cellulitis, and increased susceptibility to MRSA infections of the skin and sepsis. Congenital lymphedema is also a comorbidity of lymphatic malformations and other common syndromes (e.g. Noonan). The most common gene mutation that causes congenital lymphedema is a heterozygous inactivating mutation in the VEGFR3 gene that causes Milroy’s disease. While VEGFR3 has been widely studied as the main receptor that induces lymphangiogenesis, virtually nothing is known about how VEGFR3 regulates physiological functions of the lymphatic vasculature. Thus, the pathogenesis of Milroy’s disease remains unknown, prohibiting the development of drug therapies. Patients with congenital mutations in VEGFR3 have lower leg lymphedema and upon lymphoscintigraphy imaging it is revealed that their lymphatic vessels are unable to absorb any tracer from the interstitium. Here, we have developed a mouse model in which the VEGFR3 gene can be deleted specifically from the lymphatic vasculature to understand its physiological functions. Our preliminary data show that loss of VEGFR3 negatively affects the ability of lymphatic capillaries to remodel their continuous cell-cell junctions, reminiscent of zippers, into discontinuous wide-open junctions called buttons. Junctional remodeling in the lymphatic capillaries is a relatively new biological process that is poorly understood but relies on the adherens junction protein, VE-cadherin, in which our laboratory has expertise. Importantly, the button junctions are thought to enable fluid absorption from the interstitium. Our preliminary data identify VEGFR3 as a novel regulator of lymphatic capillary junction remodeling to form button junctions. We will combine this mouse model with cell culture and physiological approaches to investigate the role of VEGFR3 in the lymphatic vasculature in the following specific aims. In Aim 1, we will assess the ability of lymphatic capillaries to remodel their junctions in the absence of VEGFR3 at various timepoints after birth. We will also investigate whether VEGFR3 is required not only for button junction formation, but also for the lifelong maintenance of these special junctions. Lymph flow will be assessed in vivo to determine how the loss of button junctions affects physiological interstitial fluid absorption. In Aim 2, we will investigate the downstream cell signals that regulate button junction formation and identify the signaling pathways involved using a variety of approaches. The completion of these aims will identify a new signaling pathway by which VEGFR3 controls lymphatic junction remodeling to enable interstitial fluid absorption by the lymphatic capillaries. This work will significantly impact patients with congenital lymphedema by providing mechanistic insight into the pathogenesis of the disease, opening the door to developing pharmacological treatments.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTRACT As the most common cause of dementia, Alzheimer’s disease (AD) is pathologically defined by amyloid beta (Aβ) deposition in senile plaques and tau aggregation in neurofibrillary tangles (NFTs). In addition to amyloidosis and tauopathy, demyelination is also a consistent, yet often overlooked, feature of AD. Notably, myelin loss is detected even at early stages of disease with decreases observed in patients with mild cognitive impairment (MCI). This implicates that dysregulation of the oligodendrocyte cell population, the brain’s myelin- producing cells, may be a critical factor in AD pathophysiology. Several recent studies from multiple groups consistently identified transcriptional alterations in myelination networks as a key feature of AD, underscoring the great need to elucidate disease-related alterations in the oligodendrocyte population to provide novel insights into AD pathophysiology. Of particular relevance, tau accumulation is associated with loss of white matter integrity in both human patients and mouse models of tauopathy. White matter abnormalities have even been detected in cognitively-normal carriers of the apolipoprotein E ε4 (APOE ε4) genotype, a population at high risk of developing AD. Given that ApoE4 has been shown to potentiate tau toxicity and ischemia-induced white matter damage in mice, these findings may indicate that tau burden and ApoE4 converge to drive white matter abnormalities in AD. Considering that oligodendrocyte progenitor cells (OPCs) respond to white matter damage by migrating to the site of injury and differentiating into myelinating oligodendrocytes to repair the lesion, a key question is why OPCs and/or oligodendrocytes fail to correct white matter abnormalities in the presence of abnormal forms of tau and/or ApoE4. A recent study found that OPCs in both postmortem brain and a mouse model of AD exhibited markers of cellular senescence, with pharmacologic removal of senescent OPCs alleviating inflammation and cognitive defects in mice. These results provide compelling evidence that OPC dysfunction may actually contribute to and exacerbate disease progression in AD. As such, the current study will investigate the impact of tau pathology and APOE genotype on abnormalities in OPCs and oligodendrocytes, including remyelination ability. In addition, given that deletion of the ApoE receptor, Lrp1, from OPCs provided neuroprotection, stimulated myelin repair and reduced inflammation in mouse models of demyelination, we will evaluate the protective effect of Lrp1 deficiency in OPCs in the context of tauopathy, as well as the role in exacerbation of tauopathy in the presence of ApoE4. Collectively, the current project will identify key functional and transcriptional alterations observed in the OPC/oligodendrocyte population in response to tauopathy and ApoE4, and determine whether loss of Lrp1 in OPCs mitigates ApoE4-mediated exacerbation of tau pathology.

NIH Research Projects · FY 2026 · 2022-06

BIN1, the most significant late-onset Alzheimer’s disease (LOAD) susceptibility locus identified via GWAS, encodes an adaptor protein that regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. BIN1 can directly bind to tau, leading to the suggestion that BIN1 might influence AD tangle pathology. However, we and others have failed to find evidence directly linking cytosolic BIN1·tau interaction to AD risk. In contrast, compelling in vitro evidence suggests that BIN1’s function in membrane dynamics limits pathogenic tau seed uptake and influences tau release. This indicates that neuronal BIN1 might regulate tau pathology propagation. In vivo evidence to support this notion is still lacking. In order to elucidate how BIN1 function relates to disease risk for AD, it is imperative to better understand BIN1’s role in tau pathogenesis and disease progression using appropriate animal models. Our preliminary characterization of tau pathogenesis in Bin1-cKO mice reveals a complex picture: the loss of BIN1 expression in tau transgenic mice exacerbated tau pathology in the spinal cord, accelerated disease progression, and caused early death. Intriguingly, BIN1 loss also attenuated brain atrophy and protected the hippocampus from neuroinflammation, synapse, and neuronal loss, thus, profoundly reducing tau neuropathology in select regions. These intriguing findings need to be extended because of their direct clinical implications. Our central hypothesis is that BIN1 exerts its function as a risk factor by modulating tau pathophysiology in a region-specific manner. Since BIN1 is a potential target for future therapies, the overall objective of this investigation is to characterize BIN1 modulation of tau neuropathology in vivo and gain molecular insights into region-specific BIN1 functions. The goal of Aim 1 is to generate cell-type-specific inducible Bin1-cKO using CamK- and PLP-CreERT-drivers, characterize tau pathogenesis using in vivo longitudinal MR imaging and detailed neuropathology and test the hypothesis that BIN1 expression modulates tau neuropathology in select brain regions. Aim 2 studies will apply complementary stereotaxic injection approaches to directly test the hypothesis that BIN1 exerts a region-specific influence on neuron-to-neuron tau spread or influences uptake and pathology propagation via mutant tau template interaction. Aim 3 studies will perform molecular analyses through bulk and digital spatial transcriptomic strategies to map cell-autonomous and non-cell-autonomous disease-related gene expression changes and elucidate functional pathways involved in BIN1-mediated region-specific pathology modulation. This timely and unique proposal is highly innovative. This investigation using multiple Bin1-cKO mice represents the most direct in vivo approach to rigorously investigate BIN1’s involvement in the biological pathways of tau neuropathology. We believe that the successful completion of the proposed investigation will fill significant gaps in our understanding of BIN1 as a risk factor for LOAD and guide future functional characterizations of molecular pathways and pathogenic mechanisms regulated by this major LOAD risk gene.

NIH Research Projects · FY 2025 · 2022-05

ABSTRACT Extracellular histones are nuclear proteins released to the extracellular environment during tissue destruction or injury. Emerging evidence implicates them as danger associated molecules with immunostimulatory capability. The receptor-signaling mechanisms responsible for their tissue or cell-specific effects are poorly understood. In recent studies, we detected elevated plasma levels of histones in burn patients as well as animals. Administration of histones caused microvascular leakage and endothelial hyperpermeability-characteristic pathology underlying multiple organ dysfunction following burns, whereas histone inhibitors attenuated burn-induced barrier leakage. Moreover, we obtained novel evidence for the critical role of C-type lectin receptor 2d (Clec2d)-mediated tyrosine kinase signaling in endothelial response to histones. Built on these intriguing findings, this study will characterize the release, pathophysiological function, and molecular mechanisms of histones as an important contributor to burn-induced endothelial barrier injury in edema-prone tissues, including lungs and gut/mesenteric microvessels. We propose three aims: Aim 1 to characterize circulating histones in burn patients and animals correlated with organ dysfunction; Aim 2 to determine the causal effects of histones on microvascular hyperpermeability during burns; Aim 3 to explore the molecular mechanisms by which histones induce endothelial barrier breakdown. The specific mechanistic hypothesis to be tested is that following thermal destruction of tissues, injured cells release histones into the circulation where they directly interact with the vascular endothelium by binding to Clec2d and activating downstream intracellular signaling mediated by Syk/Src-FAK; these tyrosine kinases phosphorylate proteins that constitute cell-cell junctions and cell-matrix focal adhesions, thereby triggering their conformational changes and leading to increased permeability. This novel pathway will be tested in innovative experiments that incorporate newly developed imaging techniques and molecular assays into a comparative analysis of burn patients, human organs, and animal/cell models. Through this translational study, we expect to gain new insights that will not only shift the current paradigms in vascular endothelial cell biology, but also fill the gaps of knowledge in understanding burn pathophysiology. Identification of circulating histones as a key mediator of burn-induced tissue/organ injury may lead to the development of new diagnostics and therapies for thermal trauma.

NIH Research Projects · FY 2025 · 2022-03

Project Summary Cardiopulmonary arrest (CA) is a major cause of death/disability in the U.S. with poor prognosis and survival rates. The current CA therapeutic challenges are physiologically complex because they involved hypoperfusion [decreased cerebral blood flow, (CBF)], neuroinflammation, and mitochondrial dysfunction. Our long-term goal is to identify these complex regulatory elements that ultimately control neuronal viability. In our pilot study, we discovered that novel serum/glucocorticoid-regulated kinase 1 (SGK1) is highly expressed in brain NEURONS that are susceptible to ischemia (e.g., hippocampus and cortex). Inhibition of SGK1 via GSK 650394 (specific inhibitor) alleviated CA-induced hypoperfusion, neuroinflammation, mitochondrial deficits, neuronal cell death, and learning/memory deficits; this suggests SGK1 may play a detrimental role during ischemia. The primary goal of this proposal is to inhibit SGK1 and utilize pharmacological (specific SGK1 inhibitor) and cell type (neuron)-specific genetic approaches (e.g., shRNA) in our well-established rodent models of CA to answer the central hypothesis: SGK1 expression is enhanced after CA, which leads to hypoperfusion, neuroinflammation, mitochondrial dysfunctional, and neurological deficits. In Aim 1, the role of SGK1 in CA-induced hypoperfusion will be investigated. How SGK1 causes CA-induced hypoperfusion will be determined via two-photon microscopy and laser speckle contrast imaging (Aim 1a and 1c). Furthermore, we will identify potential vasoactive mediators that contribute to SGK1-mediated hypoperfusion using PCR, capillary-based immunoassay, and ELISA (Aim 1b). In Aim 2, we will determine if SGK is responsible for neuroinflammation and mitochondrial dysfunction after CA by exploring three objectives. First, how SGK1 affects microglia activation/polarization and astrogliosis following CA, which will be investigated via brain histology and flow cytometry (Aim 2a). Second, inhibition of SGK1 alleviated CA-induced neuroinflammation will be analyzed via protein chip assay (Aim 2b). Third, the harmful effects of SGK1 on mitochondrial ion homeostasis and energetics will be studied by Seahorse respirometry and microspectrofluorometry, respectively (Aim 2c and 2d). In Aim 3, we will evaluate the therapeutic potential of the SGK1 inhibitor against CA-induced neuronal cell death and neurological deficits. Utilizing brain histology (Cresyl violet and Fluoro-Jade C staining) (Aim 3a) and behavioral trials (Y-maze and novel object recognition test) (Aim 3b), the role of SGK1 in neurological deficits will be determined. Successful completion of the proposed study will reveal the fundamental roles of SGK1 in neuronal survival/death in cerebral ischemia-related diseases. Since the FDA has approved over 46 kinase-related drugs for the treatment of various diseases, our study will be promptly translated into human clinical trials for the patients suffering from CA.

NIH Research Projects · FY 2026 · 2022-02

This program is designed to train pre- and postdoctoral scientists in basic and translational research, focusing on vascular inflammation and associated disease or injury in the heart, brain, lung, kidney, gut, and placenta. As all tissues are connected via blood vessels and lymphatics, inflammatory responses in the circulation play a central role in the onset and progression of multiple organ dysfunction and pathologies, including hypertension, atherosclerosis, arrythmia, myocardium infarction, acute respiratory distress syndrome, stroke, preeclampsia, neurodegenerative disorder, traumatic brain injury, and sepsis. The recent discovery of vasculitis as a leading cause of mortality and morbidity in patients with pneumonia and sepsis further accentuates the need for future research to decipher molecular/cellular mechanisms and identify diagnostic/therapeutic targets for vascular inflammation. The goal of this program is to provide comprehensive training in research focusing on the molecular and cellular basis of inflammation and related organ injury, taking advantage of our faculty's nationally recognized expertise in inflammation research and vascular biology. The mentoring faculty consist of 20 mentors from 7 departments across 6 inter-departmental programs or research centers, 7 of whom hold MD/PhD degrees and 4 are practicing physicians; all have been funded by the NIH and have extensive experience in mentoring at both pre- and postdoctoral levels. The group has already established a close collaborative relationship in research and training, evidenced by co-mentorship for graduate students, co-authorship in numerous publications and presentations, and joint effort in grant applications. The proximity of their laboratories, along with the centralized administrative support provided by the department chaired by the program director, further enables close mentor-mentee interactions. Trainees will be selected from a large pool of PhD candidates and postgraduates in basic science programs, as well as 14 medical residency/fellowship programs related to cardiovascular sciences. The program design includes a comprehensive set of training modalities, featuring a rigorous curriculum composed of didactic courses and workshops to build knowledge and competency, an intensive research project emphasizing hands-on experience and critical/innovative thinking, and a personalized development plan to equip the trainees with not only workplace survival skills but also the vision and capability to lead independent research. Trainees will be immersed in a highly collaborative environment supported by substantial institutional resources committed to the Heart Institute, Neuroscience Institute, full-spectrum core services, bridge funding for faculty mentors, and tuition waiver and stipend supplement for trainees. In addition, the program offers several unique opportunities for trainees to learn techniques and experimental approaches that are not commonly available elsewhere, including 3/4D intravital microscopic/molecular imaging in the blood and lymph microvasculature, and translational studies using intact, functionally viable human organs.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Neuroinflammation is a key component to the establishment and progression of neurodegenerative diseases including Alzheimer’s Disease (AD). We recently identified a novel pathway called LC3-associated endocytosis (LANDO) and found it is important for mitigating neuroinflammation and neurodegeneration in a model of AD. We have robust preliminary evidence demonstrating that LANDO functions to suppress inflammatory signaling in microglia, the resident innate immune cells in the brain. Activation of LANDO facilitates the recycling of receptors that recognize β-amyloid, a contributor to AD pathology. Abrogation of LANDO results in a severe exacerbation of all markers of AD including not only β-amyloid deposition, but increased tau pathology, neuronal loss, and memory impairment. Furthermore, targeting inflammatory signaling in LANDO-deficiency is able to almost fully inhibit neuronal death while restoring microglial function and improving memory. However, the mechanisms that control LANDO in microglia and ultimately link LANDO to inflammatory signaling are unknown. We provide convincing evidence that components of retromer machinery including VPS35 and Rab11b are essential for LANDO, however are dispensable for related pathways including autophagy and LC3-associated phagocytosis. Additionally, we provide evidence that suggests LANDO alters inflammatory activation through restriction of inflammasome assembly and decreases pro-inflammatory reactive oxygen species levels. We further provide data that suggests abrogation of upstream LANDO activation and its role in inflammatory mechanisms leads to diverse programs of neuronal cell death including putative roles for the necroptotic machinery in both death and inflammation. We propose to use a variety of novel animal models and assays we have established to evaluate LANDO regulation, inflammation, and neuronal death at both the molecular and physiological levels in AD. These studies will provide new opportunities for the manipulation and development of therapeutic methodologies for AD in addition to increasing our understanding of this complex, multifaceted biological system.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY/ABSTRACT Sepsis is a critical illness arising from dysregulated host response to infection where invading pathogens elicit an adverse systemic inflammatory response that affects multiple organs and tissues. Currently, there are limited therapies that effectively treat this disease. The overarching goal of our work is to identify new molecular targets that can potentially serve as diagnostics or therapeutics for prevention and treatment of sepsis. This project focuses on glycocalyx shedding as not merely a consequence, but a critical cause, of inflammatory injury. Specifically, we hypothesize that bacterial infection promotes disintegrin metalloprotease (ADAM) upregulation and activity to shed glycocalyx molecules on endothelial surface and release their fragments into the circulation, which act as inflammatory signals to mediate microcirculatory dysfunction and barrier leakage by triggering endothelial cytoskeleton-junction responses. This novel concept will be tested by completing two aims: Aim 1 to characterize the molecular property of glycocalyx shedding products and function in microvascular inflammation during sepsis; Aim 2 to elucidate the molecular mechanisms of endothelial glycocalyx shedding and barrier injury. We propose a multifaceted approach based on an innovative design that integrates peptidomics, proteomics and nanotechnology with multispectral photoacoustic tomography, super-resolution confocal and 3D intravital microscopic imaging. Functionally viable human lungs and microvessels serve as the primary models, which are complemented by animal models and cell experiments. Microvascular barrier structure and function will be examined in-depth at the organ, tissue and cell levels under pathophysiologically relevant conditions of bacterial infection. We expect to gain novel insights that will not only fill the knowledge gaps in understanding the molecular mechanisms of septic injury, but also contribute to the development of effective therapeutics against infectious diseases. The proposed human organ studies further highlight the translational values of our work.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The long-term objective of this application is to understand whether the basement membrane (BM), the non-cellular component of the neurovascular unit, is involved in the pathogenesis of Alzheimer’s disease and can be targeted to treat Alzheimer’s disease and Alzheimer’s disease-related dementias. This is consistent with the mission of NIA. This proposal aims to investigate the biological functions of pericytic laminin in: (1) neurovascular function, including blood brain barrier (BBB) integrity, cerebral blood flow (CBF) and brain influx/efflux function, and (2) neuronal survival/function. In Aim 1, the function of pericytic laminin in BBB integrity will be investigated. First, whether and to what extent loss of pericytic laminin affects BBB integrity will be investigated using FITC-Dextrans of various molecular weights (Aim 1A). Next, the molecular mechanism underlying loss of pericytic laminin-induced BBB breakdown will be investigated, with a focus on changes in paracellular and transcellular transport in endothelial cells (Aim 1B). Furthermore, the receptors that mediate pericytic laminin’s effect in endothelial cells will be identified and examined (Aim 1C). In Aim 2, the function of pericytic laminin in CBF will be investigated. First, how loss of pericytic laminin affects CBF will be investigated using quantitative autoradiography and two-photon imaging (Aim 2A). Next, whether the reduced CBF is caused by pericyte loss/degeneration will be investigated in vitro and in vivo (Aim 2B). Furthermore, the receptors that mediate pericytic laminin’s effect in pericytes will be identified and examined (Aim 2C). In Aim 3, the role of pericytic laminin in brain influx/efflux function will be investigated. First, whether and how loss of pericytic laminin affects brain influx/efflux function will be investigated by influx/efflux assays using various fluorescently labeled macromolecules (Aim 3A). Next, whether the impaired influx/efflux function is due to BM damage and how loss of pericytic laminin affects BM composition/structure will be explored (Aim 3B). In Aim 4, the function of pericytic laminin in neuronal injury/neurodegeneration will be investigated. In this aim, whether loss of pericytic laminin leads to neuronal injury/neurodegeneration will be investigated at biochemical, structural, and functional levels. In addition, the age at which neuronal injury/neurodegeneration occurs will be determined and compared to that at which neurovascular dysfunction occurs. Successful completion of this study will elucidate the fundamental roles of pericytic laminin in neurovascular function and neuronal survival/function, and identify novel molecular targets with therapeutic potential in Alzheimer’s disease and Alzheimer’s disease-related dementias. In addition, this proposal may also lead to the generation of an innovative mouse model for neurodegeneration and open doors for new research.

NIH Research Projects · FY 2024 · 2021-09

Recent studies have reported widespread transcription of mammalian enhancers into noncoding RNA transcripts in a stimulus-dependent manner. Growing evidence shows that these enhancer RNAs (eRNAs) have essential roles in orchestrating higher-order chromatin interactions to facilitate gene expression and phenotypic outcomes during development and disease. As a result, eRNAs are emerging as an important component of the gene regulatory machinery. Due to their very recent discovery, the expression and roles of eRNAs in stroke is virtually unknown. Recently, we applied a combination of genome-wide RNA-seq and genome-wide enhancer mapping using ChIP-seq to identify a number of stroke-induced eRNAs at multiple time-points of reperfusion in the mouse cerebral cortex. Our preliminary data confirmed enhancer activity of the genomic loci encoding the eRNAs, showed that the eRNAs are localized to the chromatin, and revealed an important role for one such eRNA in modulating post-stroke brain damage and gene expression. The molecular interactions, functional mechanisms and sex-dependent effects of eRNAs on the post-stroke pathophysiology are unexplored. In the current project, we build upon our preliminary data to evaluate eRNA functionality in-depth during stroke in the adult mouse cortex. Specific Aim 1 will use a combination of crosslinking-immunoprecipitation, fluorescence in situ hybridization and transcriptional-state analysis to determine the molecular targets of the eRNAs as a function of post-stroke reperfusion time in the mouse cortex. Specific Aim 2 will employ eRNA loss-of-function in vivo followed by cellular, physiological, pathological and neurological analyses to evaluate the role of the eRNA in propagating the post-stroke pathophysiology. Specific Aim 3 will evaluate the molecular targets and pathophysiological effects of the eRNA in the female mouse cortex. Together, this work will illuminate the significance of eRNAs in the cerebral cortex and may reveal novel gene regulatory relationships in stroke. This work will pave the way for future studies exploring the therapeutic manipulation of eRNAs to improve post-stroke outcomes.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Patients with congenital immunodeficiencies, such as combined immunodeficiencies (CID) with partial recombinase activating gene (RAG) deficiency (pRD) are highly vulnerable to chronic infections and refractory autoimmune disorders. RAG1/2 are key to creating and censoring the B cell receptor diversity. In case of pRD, developing B cells that are naturally autoreactive may remain reactive to self in the periphery and be unable to mount efficient antibody responses. This results in chronic antigen exposure that can activate T and B cells. We propose to focus on two specific cell populations: hyperactive T follicular helper (Tfh) cells and innate-like extrafollicular polyreactive B cells, as markers of autoimmunity. The latter resemble age-associated B cells (ABCs), which accumulate with infections and with age. Normally, ABCs are highly sensitive to innate immune stimulation by microbes and inflammatory cytokines and play a key role in controlling viral infections by producing protective antibodies. After the infection resolves, ABC numbers contract markedly. However, with chronic infections, ABCs or ABC-like cells expand, persist and produce antibodies that are less protective against microbes and more reactive to self, especially in individuals with particular genetic immunodeficiencies. Sustained expansion of polyreactive ABCs parallels microbial/antigen load (toll-like receptor stimulation) and expansion of Tfh cells, which secrete the inflammatory cytokines, interferon gamma (IFNand interleukin 21 (IL-21). It is unclear which stimuli and cell signaling pathways are dominant in promoting ABC autoreactivity in pRD or other CID s. Our long-term goal is to understand how autoreactive ABCs emerge in CID in order to develop effective immune modulatory treatments. We hypothesize that the susceptibility to infections of patients with pRD results in increased, continual microbial/antigen presence and chronic low-grade inflammation throughout the body. In response, Tfh cells secrete inflammatory cytokines abundantly (IFNand IL-21), which together with chronic microbial stimulation induces ABC-like cells to expand and become autoantibody-secreting cells and present autoantigens to Tfh cells, which sustains them. Thus, ABC-like cell abundance and autoreactivity is perpetuated in pRD. Our specific aims are to 1) identify likely drivers of ABC- like cells in pRD patients and 2) dissect mechanisms contributing to ABC-like cell generation and persistence using pRD mouse models. Our innovative research strategies include studying an international cohort of pRD patients in parallel with using novel mouse models, exposing mice to varying levels of microbes, and correlating gene expression changes with ABC developmental characteristics and autoimmune severity of patients. Our proposed study is significant because it will likely illuminate how dysfunctional ABC-like cells develop in persons susceptible to chronic infections and lead to therapies that modify specific B cell signaling pathways to improve humoral immunity and reduce autoimmunity in pRD and other CID.

NIH Research Projects · FY 2024 · 2021-09

Project Summary We will design and develop a molecular simulation data management system, we call P2DMS, to analyze large dis- tributed molecular dynamics (MD) simulation data. The salient features of the system include: (1) a push-based local query engine design that handles data in a batch processing manner and processes many queries at the same time; (2) optimized MD analytics tools using modern many-core hardware such as GPUs; and (3) efficient management and access to distributed data over wide area networks, which is quite common for large scale MD simulations. This will be done by building a data analysis layer on top of state-of-the-art distributed big data management systems. The out- come of this project will not only improve the efficiency of MD data processing, but also enable new knowledge discov- ery that is currently regarded difficult or infeasible. In particular, we will integrate the P2DMS program into existing MD simulation packages, and validate the new design with important real-world biological and MD methodological prob- lems. In particular, we will (1) model the structure-function relationships of how the spike protein of SARS-CoV-2 inter- acts with the human angiotensin converting enzyme 2 (ACE2) receptor; and (2) enhance the performance of a recently developed parameter optimization software for active control of MD simulations.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Alzheimer`s disease (AD) is a progressive degenerative disease of the brain, a dementing illness associated with early neurovascular changes and the accumulation of misfolded amyloid-β (Aβ) and tau in the brain. At present, no effective treatment is available to slow or halt the progression of AD. Hence, uncovering novel mechanisms that govern AD pathogenesis may advance the development of more effective therapeutic strategies to treat this devastating disease. Mounting evidence suggests that the accumulation and aggregation of Ab in brain parenchyma and cerebral blood vessels (CBVs) is a key event leading to other AD-related pathologies. Kinetic studies in patients with sporadic AD indicate that faulty Aβ clearance, rather than Aβ overproduction, is critical for accumulation and aggregation of Aβ in brain. However, the molecular underpinnings of such Aβ accumulation remain poorly understood. Our preliminary studies indicate that heparan sulfate (HS), a type of sulfated polysaccharide that critically mediates cell-cell and cell-matrix interaction and signaling, is strongly reduced in CBVs of AD patients. In this application, we will test our novel hypothesis that HS expressed in CBVs normally facilitates the clearance of Ab out of the brain and that such function is disrupted in AD, leading to impaired Ab clearance. Mechanistically, we hypothesize that HS maintains CBV integrity, functions as a co-receptor in LRP1-mediated Ab clearance and facilitates perivascular Ab elimination. We will pursue the following 3 specific aims to rigorously test our hypothesis: 1. Elucidate the roles of brain endothelial cell (bEC) HS in Ab clearance and test the hypothesis that increasing bEC-HS expression normalizes Ab clearance to mitigate AD pathogenesis. 2. Delineate the molecular mechanisms underlying the roles of bEC-HS in brain Ab clearance and AD pathogenesis. 3. Elucidate the roles of brain vascular smooth muscle cell (bVSMC)- HS in brain Ab clearance and AD pathogenesis. These proposed studies exploit both novel and established genetic, cellular, scRNA-seq and biochemical approaches in conjunction with human AD specimen and AD mouse models. The results of this study are expected to illuminate HS expressed in CBVs serves as a key molecule to mediate brain Ab clearance and decreased CVS-HS expression exacerbates AD, and will provide in vivo evidence for the proof of principle that increasing bEC-HS is an effective intervention to mitigate AD pathogenesis.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract This study will test the hypotheses that: (a) increased gut permeability due to loss of mucus barrier accelerates aging-related cognitive decline and AD pathology, and (b) a unique heat-killed human-origin probiotic (Lactobacillus paracasei D3-5 [LpD3-5]) and its lipoteichoic acid (LTA) restores mucin production to reduce gut leakage and thereby ameliorate cognitive decline and AD pathology. Our hypotheses are based on multiple lines of emerging evidence, including our preliminary data indicating that: (i) Chronic inflammation begins several years before cognitive decline/AD appear in humans and mice; (ii) Increased gut permeability and reduced mucus barrier are linked with elevated inflammation in gut and brain, cognitive decline, and AD markers in older and AD mice; (iii) A unique human-origin heat-killed probiotic LpD3-5 reduces gut permeability and inflammation in the gut and brain of older mice by increasing mucin production and goblet cell numbers, and shows promising improvements in cognition; (iv) A specific LTA from the cell wall of LpD3-5 increases both goblet cell numbers and mucin production by activating toll-like receptor 2 (TLR2) signaling, which in turn reduces gut permeability and inflammation; and (v) Mucin-stimulating effects of LTA from LpD3-5 are unique, strain-dependent, and possibly due to variations in D-alanyl modification. These findings raise several important questions: (a) whether increased gut permeability due to loss of mucus barrier accelerates aging-related cognitive decline and AD pathology, and whether LpD3-5 therapy can reverse these changes; (b) how LpD3-5 and its LTA increase goblet cell numbers and thus mucin production, which in turn reduces gut permeability; and (c) why LTA from LpD3-5 differs in its mucin-promoting activity between two Lactobacillus paracasei (Lp) strains. To address these important gaps in the current state of knowledge, in Aim 1, we will define the causative role of elevated gut permeability on the onset and severity of cognitive decline/AD and its reversal by LpD3-5, using both normal aging and AD (APP/PS1) mouse models. In Aim 2, we will determine whether LpD3-5 and its LTA promote differentiation of iSCs into a goblet cell lineage in mice, to define the mechanism by which they increase goblet cell numbers in older and AD gut. In Aim 3, we will examine strain-specific D-alanyl-modification on LTAs using NMR structural analyses, to define the differences in their ability to promote mucin production via activating TLR2/Muc2 axis in vitro. Outcomes of these studies are expected to provide, for the first time, direct evidence that increased gut permeability due to loss of the mucus barrier accelerates both aging-related cognitive decline and AD, and that a unique human-origin probiotic therapy can reverse them. This work could inform a new paradigm to connect aging and AD by means of increased gut permeability as a common mechanism, and open opportunities for rational design of synthetic mimetics of LTAs to reduce gut permeability, cognitive decline, and AD – debilitating public health problems of older adults.

NIH Research Projects · FY 2026 · 2021-06

PROJECT SUMMARY Compared to their urban counterparts, rural family dementia caregivers (CGs) face increased vulnerability to insomnia and related health concerns (stress, inflammation, depression, anxiety, cognitive disturbance). Cognitive behavioral treatment for insomnia (CBT-I) holds promise for improving insomnia and these related concerns, but is difficult to access in rural areas. Our team developed brief telehealth CBT-I (tele bCBT-I) tailored for CGs (e.g., includes stress management/problem solving) that improved sleep, arousal, mood, cognition and inflammation (small to large effects). While telehealth improves accessibility, it is still burdensome for CGs due to inflexible scheduling and scarcity of trained therapists. Thus, more research is needed. Web delivery would increase access and web CBT-I is efficacious in non-CG adults, but has not been tested in rural CGs. Using the NIH Stage Model flexible framework and Medical Research Council recommendations, we developed NiteCAPP (web translation of our tele bCBT-I protocol). Stage IA/B validation and testing show high feasibility and acceptability, and improvements in sleep, arousal, mood, burden and cognition in a single arm pilot in rural CGs (n=5). The proposed trial is the next logical step - Stage II testing in an RCT (n=100) to establish efficacy and further evaluate feasibility and acceptability. The Cognitive Activation Theory of Stress provides a framework for our basic premise that CGs experience insomnia, arousal and inflammation that prompt sympathetic activation and hypothalamic-pituitary-adrenal (HPA) disruption that have downstream negative effects on health. The proposed trial tests the novel hypothesis that NiteCAPP will improve CG health, mood, burden and cognition by targeting their shared underlying mechanisms – sleep, arousal and inflammation – thereby, returning sympathetic and HPA functioning to normal. Another novel aspect of the proposed trial is inclusion of behavioral strategies to target the person with dementia’s (PWD) sleep. Outcomes will be assessed at baseline, post-treatment and two follow-ups (6 and 12 months) and include CG sleep, arousal, inflammation, health, mood, burden and cognition, and PWD sleep. The proposed study has four specific aims. Aim 1 focuses on the feasibility and acceptability of NiteCAPP and WebSHE (sleep hygiene education – active web comparator). Aims 2 and 3 examine NiteCAPP’s effects (versus WebSHE) on CG primary/mechanistic (sleep, arousal, inflammation) and secondary outcomes (health, mood, burden, cognition), respectively. Because the PWD’s sleep impacts CG sleep, Aim 4 examines NiteCAPP’s secondary effects on PWD sleep (objectively assessed). An Exploratory Aim examines the relationships between changes in CG primary and secondary outcomes, and their potential mediators/ moderators. Public Health Implications: Demonstration that rural CGs can use NiteCAPP to target sleep, arousal/stress, inflammation and related health concerns has important implications for multiple stakeholders, including rural CGs, rural PWDs, their families, clinicians and policymakers.

NIH Research Projects · FY 2025 · 2021-06

Summary The persistent gap in our understanding of ocular surface immunomodulation is a barrier for developing new therapies for inflammatory disorders that are responsible for a bulk of outpatient visits to an ophthalmologist. Previous publications from our laboratory established that the secreted Ly6/uPAR related protein-1 (SLURP1), a member of the Ly6 family of proteins is an immunomodulatory molecule at the ocular surface that: (i) is highly expressed in the corneal epithelium and secreted to the tear fluid; (ii) acts as a soluble scavenger of urokinase- type plasminogen activator (uPA); (iii) inhibits human umbilical vein endothelial cell (HUVEC) tube formation; (iv) suppresses neutrophil chemotaxis and transmigration through confluent endothelial monolayer in vitro; and (v) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production consistent with an anti-inflammatory function. Collectively, these studies identified SLURP1 as a potential therapeutic target for inflammatory disorders of the ocular surface. Here we propose to build upon these salient findings by testing the central hypothesis that ‘SLURP1 suppresses corneal angiogenic inflammation and neutrophil recruitment by regulating the TGF-β- and uPA-activities that promote NFκB-mediated production of pro-inflammatory molecules’. This hypothesis is supported by our prior publications described above, and exciting results from our unpublished preliminary studies wherein Slurp1 knockout (Slurp1X-/-) mouse corneas displayed dense corneal neovascularization and excessive neutrophil influx five days after silver nitrate cautery. Furthermore, RNA-Seq comparison of the naïve wild type and Slurp1X-/- mouse corneal transcriptomes identified key activators of angiogenic inflammation including TGF-β and NFκB pathway components to be upregulated in the absence of Slurp1, lending additional support for this hypothesis. We will test this hypothesis by employing mouse models and in vitro studies to pursue the following Specific Aims: Aim 1). Test the hypothesis that Slurp1 protects the cornea from undesirable angiogenic inflammation by suppressing unmitigated TGF-β and uPA activities that feed into NFκB pathway; Aim 2). Test the hypothesis that Slurp1 suppresses neutrophil influx into healthy corneas by promoting neutrophil maturation and clearance, and interfering with their extravasation; and Aim 3). Test the hypothesis that SLURP1 is negatively correlated with the severity of human dry eye disease and a useful therapeutic target for ocular surface inflammatory disorders. By elucidating promising new information related to the immunomodulatory functions of SLURP1, an abundantly expressed yet understudied protein, anticipated outcomes of this proposal directly address the NIH mission of ‘seeking fundamental knowledge about the nature and behavior of living systems’ and offer the potential for validating a novel therapeutic target for inflammatory disorders of the ocular surface that account for a significant burden on our healthcare system.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract The University of South Florida is uniquely qualified to conduct the activities specified in this limited competition RFA. The purpose of the RFA is to fund the continuation of the Data Coordinating Center's (DCC's) functions in support of TEDDY subject follow-up and to initiate a second nested case-control cohort. Plans to continue the DCC's functions in support of TEDDY subject follow-up include: 1) Supporting the execution of the study protocol for follow-up of TEDDY study participants 2) Receiving, managing, and analyzing data obtained from the TEDDY Clinical Centers 3) Protecting patient confidentiality at all steps in the submission and analysis of the data and ensuring the technical integrity and security of the data management systems 4) Monitoring of adherence to the research plan by conducting site visits to monitor the quality of record keeping, source documentation and the accuracy of data entry and for overseeing data quality control 5) Providing statistical support, expertise and oversight throughout the study; collaborating with the clinical investigators in the preparation of presentations and primary and secondary publications; and providing additional analyses at the request of the External Evaluation Committee (EEC) or the NIDDK 6) Providing study-wide communications, dissemination of study materials such as protocols, Manual of Operations, forms or other study documents, and development and maintenance of the web site 7) Providing training and technical assistance to the Clinical Centers in performance of the follow-up assessments; assisting in protocol implementation; and working in conjunction with the Clinical Centers and NIDDK staff to oversee all aspects of Clinical Center performance, including timeliness and quality of data and biosample submission 8) Procurement and administration of subcontracts for laboratory and repository services. Through subcontracts the DCC will also provide capitated reimbursements to the Clinical Centers for study visits and procedures, including data and biosample acquisition, and other necessary services for conduct of the study 9) Providing administrative and logistical support services for the TEDDY Study Group including preparation of publications, and organizing periodic meetings for the study group and subcommittees, workshops, and conference calls. It will also be responsible for preparing reports for and organizing meetings of an External Evaluation Committee (EEC) convened by NIDDK. 10) Ensuring the transfer of all biosamples and data to the NIDDK designated repositories. It is responsible for supporting and promoting data sharing by preparing a limited personal health information or de- identified data set in a format appropriate for data sharing for submission to a secure NIDDK data repository after publication of the primary and other study results or 12 months after completion of data collection for the primary endpoint. Plans to initiate a second nested case-control cohort will mirror the first case-control cohort and will afford the opportunity to generate data of a variety of different data types across all ages of the TEDDY study population.

NIH Research Projects · FY 2025 · 2021-04

Carbapenems, the once last-resort ß-lactam antibiotics immune to ß-lactamase hydrolysis, are now susceptible to inactivation by the so-called carbapenemases, especially the serine-based Class A ß-lactamase KPC-2 commonly found in carbapenem-resistant Enterobacteriaceae (CRE, listed as an urgent threat by CDC). Carbapenemases also threaten the future clinical utility of new carbapenems currently being developed against L,D-transpeptidases of mycobacteria and others. However, it is poorly understood how KPC-2 is able to hydrolyze nearly all ß-lactam antibiotics and continues to evade newly developed inhibitors, such as avibactam, via resistance mutations. Additionally, Class B metallo-ß-lactamases, represented by NDM-1 and VIM-2, have emerged as another problematic group of carbapenemases frequently observed in clinic, with yet few effective inhibitors. Through structure-based drug discovery, we have identified a series of phosphonate- based inhibitors of KPC-2, with the best compound displaying a binding affinity (Ki) of 20 nM and highly promising cell-based activities. Remarkably, these compounds also demonstrated low M to high nM activities against metallo-carbapenemases NDM-1 and VIM-2. Structural analysis of these inhibitors and others revealed that unique active site features of carbapenemases appear to enhance their ability to bind to small molecules. These properties enable them to hydrolyze a wide range of ß-lactam antibiotics but also make them more prone to inhibition by diverse small molecule chemotypes. In this proposal, we aim to: 1) develop low to sub- nM inhibitors against Class A carbapenemases particularly KPC-2, including dual-activity compounds with high affinity for metallo-carbapenemases as well, using structure-based design and synthesis, in vitro analysis and animal models; 2) apply mutagenesis, X-ray crystallography, NMR and MD simulation to probe the active site features, both static and dynamic, that underlie KPC-2’s broad substrate profile and unique carbapenemase activity, as well as to investigate the development of resistance against existing and new inhibitors including our own. These experiments will result in new ß-lactamase inhibitor leads for antibiotic development, while providing a deeper understanding of ß-lactamase catalysis and the evolution of resistance, to help guide future drug discovery.