University Of South Florida

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Older adults are extremely susceptible to chronic wounds such as venous leg ulcers (VLUs), diabetic foot ulcers (DFUs) and pressure ulcers (PUs). These wounds are difficult to treat and often drastic operative interventions such as amputations or free flaps with a clear loss of function are necessary. An increasingly popular approach for the treatment of chronic wounds is the use of recombinant biologics such as growth factors (GF). Elegant work by researchers over the years has shown successful healing induced by numerous GFs in various preclinical wound models developed in animals. Despite these promising results, successful translation of GFs to human patients remains limited. We have identified that this can be attributed to the presence of high levels of proteases present in human chronic wounds that result in rapid degradation of the GF rendering the therapy ineffective. Most animals models of wound healing do not exhibit high protease levels and thus therapies that tend to look promising in animals fail to translate to humans. We have developed innovative nanoparticles (NP) that not only deliver the GF at the wound site but also protect it from protease mediated degradation. Our work has shown that these NPs improve the half-life of the growth factor in human chronic wound fluid and wound healing in young diabetic mice in the presence of proteases. In this proposal, we intend to translate these promisinig NPs towards the healing of chronic wounds in the elderly. However, there are significant differences in the wound healing of aged and young animals. Therefore, in the R21 phase we propose to test the NPs in aged wounds and in the R33 stage we will focus on the preclinical development of the NPs. We will use this data to prepare an FDA application for first in human studies focused on efficacy of NPs on the healing of chronic wounds. This project will enable us to bring this promising technology closer to the bench side. As a new therapy for the treatment of difficult to heal chronic wounds, it will significantly reduce the cost and burden of chronic wounds in the health care system while improving patient outcomes and quality of life.

NIH Research Projects · FY 2026 · 2023-05

Project Summary In the anterior eye, the precorneal tear film (PCTF) acts as the interface between the ocular surface and external environment and plays a critical role in maintaining ocular surface homeostasis. In dry eye disease (DED), the PCTF becomes thinner, and destabilizes (evaporates) rapidly leading to hyperosmolarity, inflammation, and ocular surface desiccation. In 2020, the National Eye Institute (NEI) released a Notice of Special Interest (NOSI) for the Anterior Segment Initiative (ASI), “Identification and Development of New Biomarkers and Effective Methods to Diagnose Dry Eye Disease.” The notice highlighted a critical need for biomarkers and methods to diagnose DED prior to the onset of symptoms. The overall goal of this proposal is to characterize microstructural thickness variations of the tear film lipid layer (TFLL) and their association with clinical characteristics of DED. TFLL, the outmost layer of PCTF, overlies the aqueous phase, and serves as the barrier against evaporative aqueous loss, and stabilize it by facilitating the spread of its aqueous compartment and reducing surface tension. However, the exact mechanism by which the TFLL retards tear evaporation and promotes PCTF stability remains poorly understood. For instance, while most would agree that a uniform and thicker TFLL would be more protective against evaporation, and therefore prevent DED, this relationship remains controversial in the literature; resolution of this controversy forms the basis of this proposal. Under a NIH/NEI grant in 2021(R21EY033029), we constructed a novel laser source point-scanning interferometer that enables the in vivo assessment of dynamics of PCTF and related structures of TFLL with unprecedented resolution and sensitivity. Using this powerful system, we propose to address critical yet previously unexplored and often inconsistent associations between TFLL and examination findings of PCTF. The central hypothesis of the proposed research is that thickness variations in the microstructure of TFLL are associated with clinical characteristics of DED. We will test this hypothesis in concurrent Specific Aims: Aim 1: Verify and quantify the inversely relationship between TFLL thickness and PCTF evaporation rate. We hypothesize that TFLL thickness is inversely proportional to PCTF evaporation, with “thin” regions of the TFLL allowing excessive loss of aqueous tears. Aim 2: Quantify the impact of TFLL thickness variations on PCTF instability. We hypothesize that steep stress gradients at the interface between “thin” and “thick” regions of the TFLL cause PCTF instability. Collectively, the proposed studies will introduce two new parameters to characterize the lipid layer microstructure and correlate them with PCTF evaporation and instability, which will be tested and validated with our novel high-resolution interferometric system. With further clinical validation, these parameters will allow for early, non-invasive assessment of DED and inform the development of new therapeutics to slow or prevent the development of DED.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT Cardiovascular diseases remain the leading cause of death in humans, yet the molecular mechanisms underlying these devastating conditions have not been fully elucidated. Cardiac disease is especially common in the elderly, and as the global population ages their elevated incidence will pose a serious healthcare challenge. An important structure in heart muscle cells is the intercalated disc (ICD), which mediates the coordination of the cardiac syncytium. It functions by connecting neighboring cardiomyocytes, thereby maintaining the functional integrity of this syncytium; this is crucial to the proper contraction of the heart. Although many reports demonstrate the importance of ICDs in the organization of the myocardium, relatively little is known about how these cell-to-cell junctions transmit information between cardiac muscle cells to modulate gene expression and cardiac function. The Xin-repeat containing adaptor proteins Xinα and Xinβ, also called XIRP1 and XIRP2 respectively, were first discovered by the PI. These two proteins are located in the ICD of adult cardiomyocytes and interact with various adherens junction proteins including N-cadherin and β-catenin, supporting an essential role for them in the formation/maintenance of this structure. They also play important roles during early cardiac development and in the pathogenesis of heart disease. However, the role of the Xin proteins remains poorly studied and their specific cellular and molecular functions are largely unknown. Our recent studies of the hearts of Xin knock-out (KO) mice have identified defects in development associated with impaired cardiomyocyte proliferation. Our studies further demonstrated a physical and genetic interaction between Xin and NF2, a component of the important Hippo/YAP pathway. The Hippo-YAP pathway is a highly conserved cellular regulatory network that has been previously implicated in multiple developmental systems and disease, including the heart; however, the mechanisms of its action remain unclear and a link to the ICD is a novel and exciting new discovery. Therefore, we have designed two integrative Specific Aims to test the mechanism by which the ICD protein Xin mediates cardiomyocyte proliferation, maturation, and regeneration. For the first Aim, we will investigate the interaction between Xin and the Hippo/YAP pathway. We will study how Xin regulates YAP activity and how the interaction between Xin and Hippo-YAP signaling regulates cardiac function and regeneration. For the second Aim, we will study how YAP/Tead1 regulates Xin transcription and test our hypothesis that Xin-YAP cross-regulation is crucial to cardiac gene expression and heart regeneration. The studies proposed here will reveal novel molecular mechanisms by which the important pathophysiological Hippo/YAP signal is modulated by the ICD protein Xin in the heart. The molecules defined in this study will become targets for therapeutic intervention in the treatment of cardiac diseases.

NIH Research Projects · FY 2026 · 2023-04

Protein-protein interactions (PPIs) are increasingly important targets for bioorganic and medicinal chemistry, given the critical role of myriad protein complexes in vital biological processes including signal transduction, cell growth and proliferation, etc. Due to enhanced stability and functional diversity, helical peptidomimetics have emerged as a promising strategy for targeting PPIs, however their application is still limited as they generally do not mimic α-helices well owing to their difference in the three-dimensional helical structures. For instance, BCL9/β-catenin and TCF/β-catenin PPIs involved in Wnt signaling pathway play an important role in embryonic development and tissue homeostasis and are linked to several types of cancers. As such, molecules disrupting either BCL9/β-catenin or TCF/β-catenin PPIs could become novel anti-cancer agents by inhibiting Wnt/β-catenin signaling. However, the successful design of helical inhibitors based on unnatural scaffold to block the PPIs is previously unknown. We have recently developed a series of unprecedented helical sulfono-γ-AApeptides that can mimic α-helical domain of proteins. Given by the significance of β−catenin/BCL9 and TCF/β-catenin PPIs, we believe that mod- ulating these PPIs could serve as an excellent opportunity to formulate the general strategy for targeting any other PPIs involving α-helices. Compared to the BCL9 and TCF peptides not exhibiting cellular activity, our preliminary studies indicated that sulfono-γ-AApeptides not only can mimic these helical domains, but also are highly cell permeable and can selectively inhibit growth of cancer cells with hyperactive Wnt/β−catenin signaling. To the best of knowledge, our findings represents the first example of helical peptidomimetics based on unnatural backbone in disrupting these PPIs. As such, our long-term goal is to develop novel helical sulfono-γ-AApeptides that serve as proteolytically stable and cell-penetrating molecular entities capable of modulating a myriad of medicinally relevant PPIs. The objective here, is to establish the design strategy of sulfono-γ-AApeptides as helical domain mimetics to disrupt β−catenin/ BCL9 and TCF/β-catenin PPIs with optimal potency. We will first identify helical sulfono-γ-AApeptides that potently disrupt β−catenin/BCL9 and TCF/β-catenin PPIs in vitro. Fol- lowing that, we will confirm molecular mechanism of lead compounds is through modulation of Wnt signaling, by using confocal spectroscopy, TOPFlash and FOPFlash assay, cellular engagement assay, and related signaling assays to assess the selectivity, specificity and potency of these sequences on the cellular level. Finally, we will use a mouse tumor model to validate the ability of the inhibitors to inhibit Wnt signaling and tumor growth in vivo. The proposed work is significant, as these studies are highly likely to lead to a new generation of therapeutic agents targeting both β−catenin/BCL9 PPI and β-catenin/TCF PPIs, which will secure the goal of inhibiting Wnt signaling. The proposed work is innovative, because our strategy of protein-domain-mimicking using helical sulfono-γ-AApeptides is completely new and can be adopted to target myriad disease-related PPIs in the future.

NIH Research Projects · FY 2026 · 2023-04

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease. Lupus nephritis is the renal involvement in SLE with predominate glomerulus damage and exhibits a wide variety of symptoms from asymptomatic proteinuria to end-stage renal disease. Lupus nephritis requires aggressive immunosuppressive therapy, however, unfortunately most of these medications are associated with severe side effects. Therefore, development of new treatment strategies is essential. The goal of this proposal is to develop a nanoparticle delivery system that enables targeted drug delivery with stable and sustained release of prednisolone at the glomeruli as a novel therapeutic approach for lupus nephritis. Collagen IV (Col4)-α3,4 and 5 are expressed in the glomerular basement membrane (GBM), which is the only site in the body that Col4 has direct contact with blood via fenestrated capillary endothelium. Liposomes are one of the most widely used carriers due to their excellent biocompatibility and non-immunogenicity, but are lack of stability in terms of leakage of the encapsulated contents. Tripolyphosphate (TPP) cross-linked chitosan nanoparticles are characterized with controlled release of the loaded contents. Consequently, we hypothesize that by coupling Col4 binding peptides on the shell of liposomes filled with TPP-chitosan nanoparticles, we would make a kidney glomerulus selective delivery system with stable and sustained release of the loaded contents. We further hypothesize that this system loaded with prednisolone exhibits highly therapeutic efficiency due to the locally sustained drug release at a high concentration, meanwhile, it significantly minimizes the systemic side effects due to the very low dose of total prednisolone administered.

NIH Research Projects · FY 2026 · 2023-04

Project Summary G protein-coupled receptors (GPCRs) can be activated by partial agonists, resulting in submaximal signaling with reduced side effects, compared to the full agonist. Increased studies have indirectly demonstrated that partial agonists stabilize intermediate conformational states between the inactive and fully activated states with a reduced heterotrimeric Gabg protein coupling activity from the fully activated state. Due to the technical hurdles in delineating GPCR conformational states and populating individual intermediate states to study them individually, a mechanistical understanding of partial agonism signaling has been challenging. By creating conformation-biased mutants, we identified five adenosine A2A receptor (A2AR) conformational states, including two inactive states (S1 and S2), two intermediate states (S3 and S4), and a fully active state (S5), using 19F nuclear magnetic resonance (NMR) spectroscopy. This result is a significant advancement to previous research. The R291A mutant predominantly accumulates the intermediate S4 state while the R291AR293A mutant populates both intermediate S3 state and the full activated state S5. This finding enables us to study the roles of these intermediates and their complexes. We will use these two mutants to examine the roles of intermediate states S3 and S4 and their interactions with G proteins and consequent signaling effects. In Aim 1, we will characterize whether and how the intermediate states S3 and S4 interact with Gasbg. These characterizations will include the study of conformational transitions and dynamics of intermediate states and the effects of Gasbg and ligands on their transitions and dynamics. In Aim 2, we will map the conformational states of the Gas and determine its intermediate states that interact with the S3 and S4 states of the A2AR. In Aim 3, we will determine if the intermediate states S3 and S4 of the A2AR induce Gasbg states that are competent for GTP hydrolysis, G protein dissociation, and contribution to submaximal signaling without the S5 state being involved. We expect to correlate conformational and dynamic characteristics of the intermediate states of the A2AR and Gas protein created from Aims 1 and 2 to the signaling efficacies of conformation-biased mutants with ligand stoichiometries, measured in Aim 3. The completion of this proposed project will advance our understanding of the roles that intermediate conformations play in GPCR signaling, lead to a conceptual innovation in understanding receptor activation beyond a simple two-state model, and potentially guide drug design based on GPCR and G protein conformational responses to ligands. Moreover, the conformation-biased mutants will guide an approach development in resolving the structures of intermediate complexes.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT The goal of this research is to address the critical barriers to communication that older and hearing-impaired listeners face in complex acoustic scenes, in which multiple competing sounds impede speech understanding. Our approach includes simultaneous behavioral and electrophysiological measures of auditory stream segregation, and importantly, we also determine the costs and benefits of hearing instruments on segregating multiple speech sources. Our research has uncovered neural mechanisms associated with age-related declines in auditory temporal processing that play a critical role in binaural and spatial hearing. Our current tools, however, lack the precision necessary to gain deeper understanding of this age-related deficit. In the present study, we propose a series of innovative approaches that aim to determine the functional boundaries of spatial hearing in listeners with age-related hearing loss (ARHL). In Aim 1, we use a well-established paradigm of auditory scene analysis to define functional, ecologically relevant boundaries of perception as they relate to individual binaural cues, and measure how these boundaries are affected by ARHL at the neural level. Aim 2 introduces novel behavioral and electrophysiological tasks that precisely measure functional boundaries between segregation and integration of competing speech. Aim 3 evaluates the costs and benefits of directional microphone technology to hearing-impaired listeners as a function of their individual spatial segregation boundaries. The present study draws from a long line of research on auditory scene analysis and introduces a novel approach to cortical tracking of speech envelope in complex scenes. This project pushes the field forward in how we measure mechanisms of stream segregation in older and hearing-impaired listeners. The results of these experiments will: (1) precisely characterize individual binaural and spatial segregation boundaries; (2) provide evidence of the neural circuitry and patterns of activity underlying the influence of spatial cues on stream segregation; (3) discover evidence for the relationship between binaural segregation boundaries, individual differences, and the effects of hearing loss and aging; and (4) investigate a relationship between individual suprathreshold hearing abilities and specific hearing aid processing technology. Whereas hearing devices currently have remarkable benefits to specific users, the present research should inspire a new push for innovative ways to address hearing challenges of an aging population. Our team is well- positioned to execute behavioral and electrophysiological measures sensitive to auditory function and intervention outcomes, and critically, to bridge these to shift the field toward individualized hearing health care.

NIH Research Projects · FY 2024 · 2023-03

The therapeutics efficacy of an anticancer treatment is often restricted by tumor fibrous tissue and tumor microenvironment. Stromal matrix barriers create a sanctuary for breast cancer, while the prolonged pro-fibrotic stimuli facilitate cancer cell growth and survival by establishing an immunosuppressive environment. Relaxin, a small peptide hormone, demonstrated dual anti-fibrotic and pro-immunogenic effects in various disease models including several solid cancers. Treatment with relaxin significantly reduced the expression of major tumor extracellular matrix components such as collagens, fibronectin, and elastin. The degradation of fibrotic tumor matrix following relaxin treatment led to reduced cancer cell drug resistance. Additionally, it was shown that relaxin can change tumor macrophage population from pro-inflammatory to pro-resolution enabling T cell- mediated cancer cell killing and macrophage phagocytosis. However, due to a short half-life in vivo, delivery of recombinant relaxin requires continuous infusion. Relaxin signals through its cognate G protein-coupled receptor RXFP1, which is expressed in tumor associated fibroblasts and infiltrating immune cells. We propose targeting the integrity of the tumor microenvironment with the first-in-class small molecule agonist of RXFP1 developed in our laboratories. The lead compound, ML290, shows high activity, oral bioavailability, in vivo stability, and excellent pharmacological properties. It is well tolerated by animals, shows no toxicity in vivo, and does not increase cancer cell proliferation and invasiveness nor does it affect extracellular matrix remodeling in healthy tissues. Our preliminary data indicate that the ML290 treatment of mice with HER2-positive breast cancer reduces tumor size and tumor fibrotic content. The overall goal of this project is to demonstrate anticancer activity of relaxin receptor agonist, ML290, in preclinical models of breast cancer, regardless of cancer subtype. We will test the anti-cancer efficacy of ML290 in primary and metastatic breast cancer models, analyze changes in tumor ECM composition, recruitment of immune cells, and genomic/proteomic response in stromal and cancer cells to treatment. We will analyze the effect of combination treatment of ML290 with immune checkpoint blockade and anti-HER2 immunotherapeutics. The pharmacological re-programing of stromal cancer microenvironment by ML290 will provide a new therapeutic approach for breast cancer suppression.

NIH Research Projects · FY 2026 · 2023-03

Project Summary Formation of vascular lumen of appropriate size, or tubulogenesis, is one of critical steps during vascular development. Many vascular diseases including venous malformations are associated with malformed or enlarged lumens. However, we still have a limited understanding of molecular events that regulate vascular lumen size. Abelson (Abl) kinase signaling regulates diverse processes during development and disease, including cytoskeletal reorganization required for cell morphogenesis, cell motility, adhesion and polarity. Abl signaling in different cell types can induce activation of Rho GTPases, which are known key regulators of lumen formation. However, the role of Abl signaling in regulating vascular lumen size has not been previously investigated. Src homology 2 domain containing E (She) protein was originally identified as a highly conserved factor, which interacted with Abl kinase. We have previously demonstrated that She is specifically expressed in embryonic vasculature in zebrafish embryos. However, its biological function is still unknown in any organism. Here we obtained preliminary data which argues that She acts as a novel regulator of vascular lumen size. Zebrafish mutant embryos deficient in she function display enlarged vascular lumen within the dorsal aorta. Similarly, human vascular endothelial cells, deficient in SHE function, form enlarged tubes. Our preliminary data further suggest that She functions as a novel regulator of Abl signaling, and argue that ABL signaling and lumen formation are misregulated in human venous malformation (VM), suggesting a potential role for ABL and SHE in human disease. We hypothesize that SHE is a novel adaptor protein which functions in the ABL kinase signaling pathway to restrict lumen size during vascular tubulogenesis. We further hypothesize that SHE overexpression may reduce lumen size in VM leading to a novel therapeutic approach. The following specific aims are proposed: 1) Determine the cellular and molecular mechanisms by which She regulates tubulogenesis; 2) Determine if She restricts lumen size by inhibiting Abl kinase signaling pathway; 3) Determine if She overexpression can reduce lumen size in VM. Zebrafish embryos deficient in she function will be analyzed for cellular and molecular defects in vascular tubulogenesis. Conservation of She function will be tested in human vascular endothelial cells. The role of Abl kinase signaling in tubulogenesis and its interaction with She will be analyzed using chemical inhibitors, genetic mutants and biochemical assays in zebrafish embryos and human cells. The therapeutic potential of SHE to reduce the size of vascular lumen will be analyzed in the cell culture and mouse VM model as well as in primary cells isolated from VM patients. Obtained results will identify the role for SHE and ABL signaling during normal and pathological tubulogenesis, which may lead to the development of novel strategies to treat vascular malformations.

NIH Research Projects · FY 2026 · 2023-02

CRCNS US-German Research Proposal: Quantitative and computational dissection of glutamatergic crosstalk at tripartite synapses (1) Christine R Rose, Heinrich Heine University, Düsseldorf, Germany (2) Christian Henneberger, University of Bonn, Germany (3) Ghanim Ullah, University of South Florida, Tampa, FL, USA Project Description 1 Introduction and Background Transmission at chemical synapses is the central mechanism by which information is transferred between neurons. Synaptic connections such as glutamatergic excitatory synapses are often perceived and modeled as point-to-point connections. However, there is substantial evidence that crosstalk between various glutamatergic synapses can occur when the presynaptically released glutamate is sensed not only by its direct postsynaptic partner but also by nearby synapses of the same and other neurons [4]. Notably, this phenomenon termed “glutamate spillover” not only defines the input-specificity of a given synaptic connection and its crosstalk to neighboring synapses, but is also involved in and controlled by activity-dependent plasticity [1, 7, 8]. How easily glutamate escapes from its release site and how far it spreads into the tissue depends on the morphological and molecular properties of the extracellular space (ECS) as well as on the efficacy of glutamate clearance, which primarily depends on astrocytic uptake [11, 12]. We and others have shown that the efficacy of perisynaptic glutamate uptake by astrocytes displays a remarkable heterogeneity between brain regions and, importantly, can vary drastically from one synapse to the next within a brain region [3, 7, 8]. This is in part because the morphological coverage of synapses by perisynaptic astrocyte processes (PAPs) can differ strongly between individual synapses [14]. Moreover, the Henneberger lab has recently shown that higher synaptic coverage by PAPs correlates with a higher local efficacy of glutamate uptake [3]. We have also demonstrated that in addition to being heterogeneous, astrocytic glutamate uptake and PAPs morphology both are controlled by neuronal plasticity [1]. Moreover, glutamate uptake is governed by the transporters’ stoichiometry, importing one glutamate molecule into the astrocyte by using the energy gained from co-transporting three Na+ and one proton down the electrochemical gradients, whilst also exporting one K+ [12]. While the inwardly-directed Na+ gradient is the main driving force for glutamate uptake, recent work by Rose lab and others have shown that glutamatergic activity causes local or global Na+ transients in astrocytes ([Na+]A) [15]. In the mouse hippocampus, astrocytic Na+ signals in fact arise predominately due to the activity of glutamate transporters themselves, degrading the Na+ gradient and thereby transiently weakening uptake capacity in a negative feedback-loop [15-17]. In the neocortex, glutamatergic synaptic activity in addition results in prominent Na+ influx through NMDA receptors, boosting astrocyte Na+ gradients [18]. Thus, it is increasingly appreciated that astrocytic glutamate uptake is neither static nor uniform. First, it is functionally dependent on the gradients of the transported ions which dynamically change with synaptic transmission [12]. Second, it is plastic because structural remodeling of PAPs on time scales of minutes profoundly alters perisynaptic glutamate spread [1]. Therefore, the emerging hypothesis is that the degree of glutamate spillover and, therefore, synaptic crosstalk in most brain regions are dynamically regulated and controlled at the level of the astrocytes. Furthermore, since a single astrocyte can contact thousands of synapses of various neurons, it has the potential to locally control the crosstalk of many synapses. In such a scenario, an astrocyte, or a subcellular domain of it, can coordinate crosstalk between many glutamatergic synapses on different neurons. Thereby, astrocytes and their PAPs set the spatial fidelity of glutamatergic synaptic transmission and as a consequence profoundly control neuronal signal exchange. So far, these important hypotheses remain largely untested. We will fill this gap by combining quantitative fluorescence imaging, astrocytic manipulations, and predictive computer modelling. This will be accomplished by investigating perisynaptic astrocytic Na+ gradients, the main driving force of glutamate uptake, and local mechanisms controlling them 1

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract Eleutherobin (1) is a diterpenoid marine natural product (MNP) isolated from octocorals. As a potent microtubule stabilizing agent, 1 shows growth inhibition toward cancer cell lines with potency comparable to paclitaxel but with reduced cross-resistance toward B-tubulin mutants. Currently, a sustainable supply of 1 has not been accessed through wild harvest, aquaculture or total synthesis. A synthetic biology approach toward 1 has been considered as a possible alternative, but the native pathway remains elusive. Thus, the biosynthesis of 1 provides a challenging research opportunity in need of novel and creative ideas. Recently, our group has reported the characterization of a key terpene cyclase, EcTPS1, from a producer of 1, E. caribaeorum. Furthermore, the EcTPS1 gene was found to be flanked by predicted oxidase and acylase genes on an animal chromosome. This unprecedented, putative biosynthetic gene cluster (BGC) provides a clear direction for reconstituting biosynthesis of 1. Our underlying hypothesis is that by using our characterized EcTPS1 as a starting point we can produce 1 using a combination of chemical and enzymological methods. The overall goal of this proposal will be to engineer heterologous production of precursors to 1, characterize the tailoring enzymes in the BGC and employ these in a semisynthesis of 1. This work will provide innovation in the field biochemistry by further developing tools in secondary metabolism as well as affording commodities in the form of sustainable natural product supply and novel biocatalysts. Three essential challenges toward these efforts are: 1) No synthetic biology route or other sustainable approach to a eunicellane precursor exists; 2) Installation of oxygenated functional groups by chemical synthetic means will require stereo-, regio- and chemoselective methods. 3) The tailoring enzymes of the biosynthetic pathway are biochemically challenging membrane bound proteins. These challenges will be addressed using organic synthesis and synthetic biology as outlined in the following specific aims: Aim 1) Engineering a semi-synthetic route toward eleutherobin; Subaim 1 a) Synthetic biology route to the eunicellane precursor klys implexin R; Subaim 1 b) Chemical synthesis of the eleutherobin core: Aim 2) Characterization of tailoring enzymes in the eleutherobin biosynthetic pathway; Subaim 2a) Characterization of cytochrome P450 enzymes; Subaim 2b) Characterization of acyl transferase enzymes.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Cardiovascular disease remains the leading cause of death in the United States; however, the genetic causes and molecular mechanisms underlying these medical conditions have yet to be thoroughly elucidated. As a result, the identification of new therapeutic targets for the effective treatment of these diseases is urgently required. Because the majority of the genome is actively transcribed to produce a vast number of non-coding RNA transcripts, this project is focused on determining the role of long non-coding RNAs in heart disease, which is an understudied and important area of investigation. Thousands of long non-coding RNAs (lncRNAs), which are defined as non-coding RNA transcripts greater than 200 nucleotides in length, have been found to have biological activity in humans and other organisms; they are considered novel regulatory molecules of numerous physiological and pathological processes, including those in the cardiovascular system. Our earlier RNA-seq studies identified many differentially expressed lncRNAs in the hearts of patients with ischemic cardiomyopathy. One of these lncRNAs, known as large intergenic non-coding RNA-p21 (lincRNA-p21), was previously shown to be a transcriptional target of tumor protein p53 and a novel regulator of cell proliferation and apoptosis. More recently, it has been implicated in the control of the regulation of vascular remodeling responses in atherosclerosis. However, the function of lincRNA-p21 in the heart and cardiac disease remains unknown. We propose to examine the function of lincRNA-p21 in hypertrophic cardiomyopathy and cardiac regeneration. Our preliminary data using mutant mouse lines demonstrates that lincRNA-p21 is also involved in cardiac remodeling in response to pathophysiological stress. Therefore, the overall goal of this application is to clearly define the function and molecular mechanisms of lincRNA-p21 in the heart. We propose to do this by pursuing the following Specific Aims: 1) to systematically study the in vivo function of lincRNA-p21 in the heart using gain- and loss-of-function mouse models, 2) to define the molecular mechanisms by which lincRNA- p21 regulates cardiac remodeling by testing the hypothesis that this lncRNA alters cardiac function by affecting the expression and function of Kap1/Trim28-dependent genes, and 3) to examine the therapeutic potential of lincRNA-p21 in treating cardiomyopathy using adeno-associated virus (AAV) vectors to either overexpress, or AAV/gapmers to knockdown this lncRNA, in mice with cardiomyopathy. As a result, this proposal will systematically and rigorously assess the role of lincRNA-p21 in heart disease and establish the molecular mechanisms underlying the function of this intriguing lncRNA. The information obtained from these studies are anticipated to identify important new molecular targets for the therapeutic treatment of cardiac disease.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Atrial fibrillation (AF) is the most common arrhythmia and its prevalence is rising alarmingly. It is particularly challenging to treat persistent AF and to restore normal sinus rhythm with currently available antiarrhythmics. Therefore, novel ion channel blocking modalities are needed for the development of the next generation of antiarrhythmic pharmacotherapies in persistent AF. A hallmark of remodeling in the chronically fibrillating atria is the presence of a constitutively active, parasympathetic stimulation independent acetylcholine sensitive inward rectifier potassium current (IKACh). Constitutively active IKACh acts as a background inward rectifier conductance and can contribute to the shortening of the atrial effective refractory period, and consequently to the perpetuation of AF. We demonstrated earlier that IKACh blockade with the peptidotoxin tertiapinQ, a 21-amino acid synthetic peptide blocker of IKACh originally isolated from the European honeybee venom, terminates persistent AF. We thus bioengineered, produced and characterized a novel, and potent IKACh blocking peptibody. Peptibodies are chimeras generated as fusion proteins of the fragment crystallizable (Fc) domain of the human immunoglobulin G (IgG1) with a bioactive “warhead” peptide. Peptibodies combine the biologic/therapeutic activity of a given peptide, with the stability of monoclonal antibodies and are stable and safe molecules that are emerging as viable clinical therapies. Our IKACh blocking peptibody was constructed as a fusion protein between the Fc fragment of human IgG1 and tertiapinQ linked together by an octaglycine spacer. In this application, we propose to test the hypothesis that bioengineered peptibodies designed as potent and bioactive blockers of IKACh are antiarrhythmic in persistent AF. Our goals are: 1- to delineate the structural determinants for the peptibody’s block of IKACh; 2- to determine the atrial specificity, electrophysiological safety and therapeutic potential of anti-IKACh peptibodies in a pig model of persistent AF; and 3- to bioengineer next generation peptibodies that have increased IKACh potency and specificity. Successful accomplishment of our proposal should be a major step forward in the deliberate, innovative, and rational development of much needed and effective antifibrillatory agents based on bioengineering approaches that target ion channels important in the mechanism of a major cardiac disease.

NIH Research Projects · FY 2025 · 2022-09

Project summary A major impediment to adult emergency department (ED)-based HIV/HCV screening success is that often ED patients at risk for, or later diagnosed with, HIV and HCV decline testing. However, for patients who decline HIV/HCV screening, there is no evidence-based intervention to persuade them to be tested. In response to the deficit of an intervention to increase ED HIV/HCV screening acceptance, we recently completed an R34 project during which we developed a persuasive health communication intervention (PHCI) with stakeholder assistance (ED patients, HIV/HCV counselors, and ED medical staff). In two pilot studies, we examined the efficacy of the PHCI among adult ED patients who initially declined HIV/HCV screening. In one pilot study, adult ED patients assigned to watch a control condition video were more likely to agree to be tested than those who received the PHCI in-person from an HIV/HCV counselor (n=56, 27% vs. 10%; ∆17%, ∆95% CI: 6-32%). However, in a separate pilot study, adult ED patients randomly assigned to watch a video of a physician delivering the PCHI to an ED patient were more likely to agree to be tested than those who watched the control condition video (n=60, 29% vs. 11%; ∆18%, ∆95% CI: 7-35%). These results suggest that the PHCI video might increase HIV/HCV screening acceptance more than a PHCI delivered in-person by an HIV/HCV counselor. If these results are confirmed in a larger study, the PHCI video could enable wider usage of the PHCI and facilitate greater acceptance of ED HIV/HCV screening. Unfortunately, in our R34 project we did not develop nor evaluate the PHCI among current and former people who inject drugs (PWIDs). As such, the current PHCI might be inadequate in persuading current/former PWIDs to be tested, and thus needs modification with the help of current/former PWIDs to be effective for these very high HIV/HCV-risk patients. In this R01 project, we first will modify the PHCI with input from current/former PWIDs. Next, we will conduct a randomized, controlled trial (RCT) to compare the PHCI’s efficacy when delivered by a video vs. an HIV/HCV counselor. Adult ED patients who declined HIV/HCV screening will be stratified by IDU history: (1) current/former PWIDs or (2) never/non-PWIDs. Within each of these strata, we will randomly assign participants to a PHCI delivered by (1) video (2) HIV/HCV counselor. Current/former PWIDs will receive the PHCI modified for PWIDs, whereas never/non-PWIDs will receive the non-modified, original PHCI version. For Aim 1, we will determine which delivery form of the PHCI (video or HIV/HCV counselor) results in more ED patients accepting HIV/HCV screening, independent of their history of IDU. For Aim 2, we will determine which delivery form of the PHCI results in more ED patients accepting HIV/HCV screening within each IDU history cohort (current/former PWIDs, never/non-PWIDs), and if testing acceptance is similar across IDU history cohorts. For Aim 3, we will further compare the two PHCI delivery forms through a health economics assessment, both independent of IDU history and within each IDU history cohort.

NIH Research Projects · FY 2024 · 2022-09

ABSTRACT Maternal opioid use disorder (OUD) is the leading cause of maternal mortality in the first year after delivery nationwide. OUD also contributes substantially to out-of-home placements in the child welfare system. Medication for OUD (MOUD) is the primary standard of treatment, however, access to MOUD and prenatal care is limited, siloed, and fragmented in Florida. Gaps in access to and continuity of healthcare (prenatal, postpartum, pediatric, pharmacological and behavioral health) and other services for mothers in OUD recovery lead to poor outcomes for parent, child and family. There is also insufficient data integration, due to inconsistent data collection methods or use of diagnostic codes, to identify mother-infant dyads affected by OUD that could inform optimal care at the local level. Single-site studies that integrate substance use disorder programs in pregnancy have been shown to improve neonatal and maternal outcomes. With that in mind, the long-term goal of this study is to leverage high-quality local and timely data to improve OUD outcomes before, during, and after pregnancy with an integrated care approach that can be replicated throughout the state. The objective of the proposed project is to consolidate multiple streams of public health and clinical healthcare data to analyze equitable access and outcomes for families affected by maternal OUD for use in quality improvement cycles to rapidly refine our integrated CADENCE (Continuous and Data-Driven Care) Program. Our central hypothesis is that integrated, continuous, data-driven care will improve CADENCE patient outcomes. We will test this hypothesis through the following aims: 1) create an interactive data dashboard for maternal, neonatal, and infant outcomes for pregnancies affected by OUD; 2) pilot the CADENCE program and rapidly refine using a data-driven approach; 3) determine the improvement in clinical outcomes at the program level using the data from the dashboard and assess the implementation of the CADENCE program; 3) analyze the cost of the CADENCE program and long-term costs of maintenance of the program. Upon completion of our aims, the expected outcomes include improve maternal, neonatal, and infant outcomes using an integrated care model and data-driven approach to tailor services to community and patient needs. Primary outcomes increase in maternal engagement in recovery or MOUD treatment at delivery, neonates with NOWS (neonatal withdrawal syndrome) requiring pharmacologic treatment at delivery, and infant’s age at referral to the Early Steps early intervention program. Our proposed plan is innovative as it is grounded in systems thinking, uses population-level and integrated clinical data to develop a dashboard methodology, and addresses an area of medicine with few previously published efficacy studies. This project meets the goals of the NIH as it aims to decrease racial inequities in prenatal care and increase access to prenatal care to decrease maternal and child mortality and morbidity.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Despite their small size, bacterial cells precisely synchronize cellular processes in space and time. The cell envelopes of bacteria are complex and dynamic structures that are coordinately assembled during the cell cycle. Cell wall anchored surface proteins of Gram-positive bacteria are major cell envelope components, which are secreted across the cytoplasmic membrane and covalently attached to cell wall peptidoglycan by sortase A (SrtA). Strikingly, many surface proteins contain a specific YSIRK/G-S signal peptide that targets proteins to the cross-wall during cell division. Coordinated with cell division and cell wall synthesis, cross-wall targeting promotes efficient incorporation of surface proteins to the newly synthesized cross-wall peptidoglycan; however, the mechanisms remain unknown. It has been proposed that the YSIRK/G-S signal peptide promotes localized secretion at the division septum. However, by developing a new microscopy method in our model organism of Staphylococcus aureus, we now provide evidence that in contrast to the prevailing model, the targeting does not occur during secretion, but rather is SrtA-dependent. We further discovered that cross-wall targeting is regulated by another important cell envelope component: LtaS-mediated lipoteichoic acid (LTA) synthesis and D- alanylation of teichoic acids. Intriguingly, LTA synthesis and D-alanylation regulate different biogenesis stages: LTA regulates SrtA-mediated septal anchoring whereas D-alanylation modulates cross-wall deposition. Collectively, these recent discoveries from my own lab form the foundation of my independent research program for this MIRA application. We will elucidate the distinct mechanisms by which the YSIRK/G-S signal peptide, LTA synthesis and D-alanylation spatially regulate surface protein biogenesis during the cell cycle. Successful completion of the research projects will not only reveal novel mechanisms underlying surface protein biogenesis in Gram-positive bacteria, but also uncover novel functions of LTA and D-alanylation in cell envelope assembly, providing fundamental insight into how bacterial cells precisely coordinate cell envelope assembly during growth and cell division.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Alcohol use disorder (AUD) continues to be a widespread problem with an estimated 29% of all Americans ages 12 and up meeting DSM-5 criteria for diagnosis during their lifetime (13.9% within the past 12 months; Grant et al., 2015). Although abstinence remains as the primary outcome for many treatment programs, such definitions of “recovery” remain far too narrow. Recently, NIAAA disseminated a definition of recovery aimed at addressing limitations of past research, stimulating new research, and better operationalizing recovery (NIAAA, 2020). Specifically, NIAAA defines recovery as “a process through which an individual pursues both remission from AUD and cessation from heavy drinking.” They further go on to state that “an individual may be considered recovered if both remission from AUD and cessation from heavy drinking are achieved and maintained over time.” Although this new operational definition will help to organize the existing literature and provide guidance for future research, several questions remain. First, categorization of recovery as initial, early, sustained, and stable requires further research to establish whether such thresholds are meaningful in both clinical practice and research. Second, the utility of heavy drinking thresholds to define recovery remains questionable at best. Finally, the new definition does not provide a conceptual framework for which recovery is a “process” requiring continual monitoring for clinical markers (clinical change points) that may impact recovery status. The aim of the current application is to examine the utility and validity of this new definition within the context of a novel theoretical model of AUD recovery. The proposed study will recruit participants seeking treatment for AUD from the community. Participants will complete a structured clinical interview and provide information on their current alcohol use and related behaviors. All participants will receive 12 weeks of AUD psychotherapy and complete brief assessments at the end of each treatment session and biweekly during the first 12-months post treatment. In addition, participants will complete in-person interviews at 3-month and 6- month intervals post-treatment for the duration of the study (for up to 24-54 months post treatment depending on time of enrollment). Findings from the proposed research have the potential to increase understanding of the dynamic nature of recovery and thereby improve clinical decision-making and generate future research. Specifically, our goal is to address the question of “Are the constructs of relapse, recurrence remission, or recovery useful heuristics for clinical practice and research, and if so, how?” Identification of the processes important for each type of change in clinical course may help in designing adaptive treatments that capitalize on our current knowledge of the treatment literature. For example, people may need to use different strategies (e.g., weighing pros/cons, substituting new behaviors) for different phases of recovery. To our knowledge, this application is the first to test NIAAA’s new definition and the dynamic nature of recovery, as well as cognitive, behavioral, and affective processes hypothesized to be important for initiating versus maintaining change.

NIH Research Projects · FY 2025 · 2022-09

Summary/Abstract Lithium is a first-line therapy for millions of people suffering from bipolar disorder, and is promising for inhibiting development of dementia. Experiments show that a primary mode by which Li+ alters physiological processes is by reducing activities of a surprisingly limited number of Mg2+-dependent phosphoryl-transferring enzymes, including phosphomonoesterases and protein kinases. While the (Li-independent) catalytic mechanisms of these enzymes are quite well-understood, much about the mechanistic details underlying their Li-susceptibility remain unknown. Not surprisingly, it remains a major challenge to design enzyme variants that are Li-resistant, and use them to disentangle signaling pathways associated with Li-susceptibilities of individual enzymes. Here we focus on Li+'s action on kinases, and address the following problem central to alleviating the issues raised above. Experiments on 71 human kinases show a wide range of Li-susceptibility — many are unaffected and others are affected to varying degrees. But there is no explanation for these variations. We address this gap in our understanding of Li-action by using state-of-the-art molecular mechanics (MM), quantum mechanics (QM) and QM/MM simulations, as well as mutagenesis experiments guided by bioinformatics and natural selection. Supported by experiments, we explore the overarching hypothesis that Li+ affects kinase activity by interacting directly with their catalytic sites. In Aim 1, simulations will examine how Li+ binds kinases, and how Li+ binding reduces kinase activity. Additionally, simulations will provide insights into potential allosteric effects that regulate catalytic site activity. Our biochemical, cellular and in vivo experiments in Aim2 are designed to (i) systematically examine effects of sequence differences between Li-sensitive and Li- resistant kinases, with the goal of making a Li-sensitive enzyme, GSK-3, resistant to Li+; and (ii) discover key residues that make certain kinases Li-sensitive. Experiments will also validate findings from simulations, and at the same time, simulations will provide molecular insights to interpret results from mutational experiments. Combined analysis of results from simulations and experiments will yield a Li-resistant GSK-3, which is significant because it will, for the first time, enable us to disentangle GSK-3-driven physiological effects of Li+ from those of other Li-sensitive enzymes. This study will also provide a physical basis to explain observed variations of Li-sensitivity across kinases, and these biophysical findings will serve as foundations for future efforts to make other Li-sensitive kinases resistant to Li+, and map their specific phenotypes. We expect that such efforts will improve understanding and predictions of patient responses to Li-treatments and dosages, which remains a difficult task. This will both expedite therapy and avoid exposure to side effects. Finally, this study will explore new advancements in modeling enzyme reactions and yield a validated polarizable force field for describing Li+/Mg2+ interactions with proteins. This will enable future reliable studies of Li-action on proteins not considered in this project and broaden exploration of the full range of Mg-binding proteins.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY BIN1, an adaptor protein encoded by the second most common susceptibility GWAS risk factor of late-onset AD, regulates membrane dynamics in the context of endocytosis, membrane remodeling, and synaptic vesicle release. Large-scale expression datasets have reported high-level BIN1 expression in microglia, and AD- associated BIN1 SNPs are thought to alter BIN1 expression through a microglia-specific enhancer. However, the precise functional role(s) of microglial BIN1 in regulating AD pathophysiology has not been investigated systematically. Our central hypothesis is that microglial BIN1 plays an essential role in neuroinflammatory signaling through which BIN1 influences AD pathophysiology. Our preliminary studies show that the loss of Bin1 expression in vitro (cultured microglia) and in vivo (microglia-specific cKO mice) profoundly impairs proinflammatory gene expression and the upregulation of several disease-associated microglia (DAM) genes. Our transcriptomic profiling identified BIN1 as a homeostatic microglial regulator with a non-redundant role in activating proinflammatory response upstream of Apoe, Trem2, and Tyrobp, and upstream of PU.1 and IRF1. BIN1 was predicted to regulate type 1 and 2 interferon responses in microglia in vitro and in vivo. Collectively, these findings offer important insights into microglial BIN1 function, demonstrating its significance in brain inflammatory response. The overall objective of this proposal is to explore BIN1’s role in microglia further, especially in the context of AD pathogenesis, and gain molecular insights. The goal of Aim 1 is to generate 5XFAD:Bin1 cKO mice to elucidate microglial BIN1 function in the modulation of cerebral amyloid burden and amyloid-associated pathophysiology. We will conduct detailed biochemical, molecular, and neuropathological characterization and perform transcriptomics profiling of neuroinflammation and DAM transition to understand BIN1’s role in microglial response to amyloid pathology. Aim 2 studies seek to generate PS19:Bin1 cKO mice to elucidate the involvement of microglial BIN1 function in tau pathophysiology and pathology propagation using detailed neuropathology and comprehensive biochemical, proteomics, and molecular analyses. Aim 3 studies will investigate the mechanistic role of BIN1 as a crucial regulator of early inflammatory signaling events in microglia. We will use unbiased and hypothesis-driven approaches to define the microglial BIN1 interactome and elucidate how BIN1 and its binding partners are reorganized in a context-dependent manner to facilitate immune signaling via key microglial receptors. This timely and unique proposal is highly innovative. Our strategy to use microglia-specific inducible Bin1 cKO mice represents the most direct in vivo approach to rigorously investigate how microglial BIN1 regulates AD pathophysiology and gain insights using comprehensive transcriptomics, proteomics, and interactome characterization. We believe that the successful completion of the proposed research 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 · 2022-09

Project Summary The podocyte has become a crucial focus of research and clinical efforts as a target for kidney disease interventions due to its vital role in regulating glomerular permeability and maintaining glomerular structure. Podocyte injury is pathogenetically and prognostically important in diabetic kidney disease (DKD). One of the main factors determining pathological changes in glomerular morphology and permeability is the elevation of basal intracellular calcium ([Ca2+]i) levels in podocytes, which can occur due to activation of various signaling cascades. Protease-activated receptors (PARs) are emerging as proteins of interest for their potential to modulate podocyte [Ca2+]i levels, especially under pathological conditions, such as DKD. Clinical studies have demonstrated that circulating concentrations of PAR-activating proteases are associated with DKD. Furthermore, the recent prospective OPTIMUS-5 study revealed several beneficial effects of the FDA-approved PAR1 antagonist Vorapaxar in type 2 diabetes mellitus. However, despite crucial evidence for the importance of PAR signaling pathways in podocytes in DKD, this area is still understudied. Our preliminary data demonstrate the functional presence of a PAR-GPCR-TRPC6 signaling pathway in rat and human podocytes that is increased under diabetic conditions. Consistent with these findings, we found that serine proteases promote activation of PAR1-TRPC6 cascade in podocytes from freshly isolated rat glomeruli, which triggers a rapid elevation of [Ca2+]i. Furthermore, our pilot studies have revealed that these signaling pathways are highly upregulated in a rat model of type 2 Diabetic Nephropathy (T2DN rats), similar to clinical observations in human patients. The central hypothesis of this proposal is that during the development of DKD in type 2 diabetes, when urinary thrombin and urokinase concentrations increase rapidly, overstimulation of PAR1 promotes excessive [Ca2+]i levels in podocytes through activation of TRPC6 channels, ultimately leading to cell apoptosis, development of albuminuria and glomerular damage. Thus, inhibition of PAR1 activity will mitigate podocyte damage and may be of therapeutic benefit in DKD. Several innovative approaches and unique rat models will be utilized to test the following Specific Aims: Aim 1 will test the hypothesis that PAR1 expression and its activity increase during the progression of DKD in type 2 diabetes and that this pathway contributes to the alterations in Ca2+ homeostasis in podocytes and glomerular damage; and Aim 2 will provide mechanistic insight into the activation of PAR-1 mediated signaling in podocytes and associated glomerular structure and function changes. In addition, the correlation of PAR signaling in podocytes and sex difference in the development of DKD in T2DN rats will be explored.

NIH Research Projects · FY 2025 · 2022-09

Contact PD/PI: DAVIS, IRENE S Plantar fasciitis, a repetitive strain injury, is one of the most common causes of foot pain. Ten percent of aging adults experience plantar fasciitis with 50% of the cases being disabling. Foot pain can lead to other problems such as reduced mobility, depression and prescription medication use, leading to a reduced quality of life. Relationships have been established between foot pain and weakness, reduced static and dynamic balance, and reduced walking speed in aging adults. The standard of care for plantar fasciitis is to brace the foot with foot orthoses and supportive shoes. However, chronic support of the arch has been shown to lead to intrinsic foot muscle atrophy. As plantar fasciitis is associated with this atrophy, treating it with chronic arch support only increases the risk for recurrence. In fact, there is a 50% recurrence rate and a 45.6% risk of having plantar fasciitis 10 years after the onset of symptoms in older adults. Minimal shoes are designed to allow the foot to function naturally, as if barefoot. They have already been successfully implemented in older adults for the treatment of knee osteoarthritis. Minimal shoes are highly flexible and lack the support of conventional footwear. This places greater demand on the foot muscles which promotes strengthening. Stronger foot intrinsic foot muscles have been shown to reduce the strain on the plantar fascia with each step, thereby reducing the risk of developing plantar fasciitis. Long-term Goal: To improve treatment interventions for plantar fasciitis in aging adults. Specific Aims: 1. Compare pain and physical function between the minimal footwear (MF) and the foot orthotic (FO) groups at the 3 and 6 month follow-up, 2. Compare changes in intrinsic foot muscle size and strength between MF and FO groups at the 3 and 6 month follow-up, and 3. Compare the incidence of recurrence of plantar fascial pain between the MF and FO groups at the 12 month follow-up Methods: 120 participants with plantar fasciitis (>6 mos.) will be recruited from 2 sites. They will be randomized into a minimal footwear (MF) and foot orthotic (FO) group. Baseline measures of foot muscle size and strength, static and dynamic balance, 6 min. walk test and two functional outcome questionnaires will be obtained. The MF group will receive 2 pair of minimal shoes, and the FO group will receive 2 pair of supportive shoes and prefabricated foot orthoses. The MF group will be given a 4-week program of foot strengthening and flexibility exercises, while the FO group will only receive the flexibility exercises. Both groups will be given an 8-week footwear transition program based upon their individual baseline step counts. They will return to the laboratory to repeat the baseline measures at 3 and 6 mos. Plantar fascial pain and daily step counts will be monitored over the course of the year. Comparisons in outcome measures as well as plantar fasciitis recurrence will be compared between the groups. Project Summary/Abstract Page 6

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Age-related macular degeneration (AMD) is one of the leading causes of irreversible visual dysfunction in older individuals in developed countries, resulting in loss of productivity, independence, and quality of life, as well as tremendous healthcare and economic burden. Visual dysfunction in AMD patients could be in the form of ‘‘dry’’ AMD or ‘‘wet’’ AMD or both. While some treatments are available for wet-AMD, but there is no effective treatment for geographic atrophy (GA), the advanced form of dry-AMD. Oxidative stress-induced cellular changes play a significant role in the loss of macular RPE and photoreceptors in dry-AMD. Treatments involving local and sustained delivery of molecules or genes to counteract oxidative stress-induced cellular changes could prevent RPE atrophy. Systemic or retinal delivery EPO-R76E, a modified form of EPO (with reduced erythropoietic activity) improved the function of ganglion cells and photoreceptors cells in the retina. Because of its effect in preventing cell death due to induction oxidative stress, we are especially interested in investigating the precise mechanism(s) of how RPE specific EPO-R76E interacts with other retinal cells and influences aberrant molecular pathways in controlling dry-AMD phenotype. We will interrogate the impact of EPO-R76E using two different animal models showing AMD pathology; one is associated with induction of RPE oxidative stress, and the other due to complement dysregulation. We will use recombinant adeno-associated virus (AAV) with serotype 1 to achieve sustained expression of EPO-R76E and deliver to mice eye via subretinal injection. Our first aim will test molecular mechanisms of the retinal protection by EPO-R76E using proteomics analyses of pathways and cell- specific transcriptional approaches in a mouse model of dry-AMD. Our second aim will test whether sustained expression of EPO-R76E ameliorates dry AMD phenotypes in animal models and interrogate how late in the course of retinal degeneration EPO-R76E will be effective in preventing disease symptoms. Our research will elucidate the role of EPO signaling in RPE function, retinal health, and the approach for preventive or therapeutic intervention of dry-AMD. These studies will identify novel molecular pathways for manipulating the retina and provide a new direction for managing dry-AMD.

NIH Research Projects · FY 2025 · 2022-09

Our group aims to integrate the use of voice as biomarker of health in clinical care by generating a substantial multi-institutional, ethically sourced, and diverse voice database linked to multimodal health biomarkers to fuel voice AI research and build predictive models to assist in screening, diagnosis, and treatment of a broad range of diseases. Data collection will be made possible by software through a smartphone application linked to electronic health records (EHR) and other health biomarkers such as radiomics, and genomics, and supported by federated learning technology to protect data privacy. Based on the existing literature and ongoing research in different fields of voice research, our group has identified 5 disease categories for which voice changes have been associated to specific diseases and around which we aim to center the data acquisition efforts: 1. Vocal Pathologies (Laryngeal cancers, Vocal fold paralysis, Benign laryngeal lesions) 2. Neurological and Neurodegenerative Disorders (Alzheimer’s, Parkinson’s, Stroke, ALS) 3. Mood and Psychiatric Disorders (Depression, Schizophrenia, Bipolar Disorders) 4. Respiratory disorders (Pneumonia, COPD, Heart Failure, OSA) 5. Pediatric diseases (Autism, Speech Delay) Specific Aim #1: Data Acquisition Module: - To build a multi-modal, multi-institutional, large scale, diverse and ethically sourced human voice database linked to other biomarkers of health that is AI/ML friendly to fuel voice AI research Specific Aim #2: Standard Module: - To introduce the field of acoustic biomarkers by developing new standards of acoustic and voice data collection and analysis for voice AI research. Specific Aim #3: Tool Development and optimization - To develop a software and cloud infrastructure for automated voice data collection through a smartphone application that allows non-invasive, user-friendly, high quality voice data collection while minimizing human manipulation. This will include integrated acoustic amplifiers and acoustic quality standardization. - To implement Federated Learning technology to allow analysis of multi-institutional data while minimizing data sharing and preserving patient privacy Specific Aim #4: Ethics Module - To integrate existing scholarship, tools, and guidance with development of new standard and normative insights for identifying, anticipating, addressing, and providing guidance on ethical and trustworthy issues from voice data generation and AI/ML research and development to clinical adoption and downstream health decisions and outcomes. - To develop new guidelines for consenting to voice data collection, voice data sharing and utilization in the context of voice AI technology Specific Aim # 5: Teaming Module: - To build bridges between the medical voice research world, the acoustic engineers, and the AI/ML world to promote the integration of tangible clinical application for Voice AI algorithms Specific Aim #6: Skills and Workforce Development Module - To develop a unique curriculum on voice biomarkers of health and the development, validation, and implementation for AI models that are FAIR and CARE - To create a community of voice AI researchers, especially those from underserved communities, and foster collaborations to promote application of ML for Voice Research - To engage a broad range of learners with competency assessment and mentorship

NIH Research Projects · FY 2026 · 2022-09

Project Description/ Abstract Corneal scarring is a public health problem and a very common indication for corneal transplantation. Disorganized deposition of extracellular matrix or scarring occurs after different pathological processes including corneal infections, congenital diseases and trauma. The long term goal of this proposal is to address the innovative concept that reestablishment of a unique environment or stromal niche with its unique mechanical and chemical cues is critical after injury to ameliorate scarring. Understanding the mechanisms that direct reestablishment of stromal function after injury is a potential target for therapuetic interventions. Our preliminary results support these concepts. The specific aims are to: (1) elucidate the regulation of collagen V reexpression in the reestablishment of corneal stroma function; (2) determine the role of this unique microenvironment in regulating keratocyte-fibroblast- myofibroblast differentiation; and (3) determine the roles of collagens V, in regulating latent TGF-β activation and therefore modulating matrix deposition and scarring. This work will provide critical information for the development of future medical or surgical therapies for patients with corneal scarring and will open the door for therapeutic manipulation of the corneal stroma.

NIH Research Projects · FY 2025 · 2022-08

Acute kidney injury (AKI) is associated with higher risk of developing chronic kidney disease (CKD), which is a growing health problem afflicting over 37 million US adults with cost over $80 billion every year. However, the underlying mechanisms, especially the risk factors that contribute to development into CKD for people with AKI has not been fully elucidated. Additionally , no specific therapy is available in prevention of AKI to CKD transition. Therefore, further understanding the pathophysiological mechanisms is essential for identification of new therapeutic targets for prevention of AKI to CKD transition. Decrease in GFR is a hallmark for AKI and CKD. TGF response is one of important mechanisms that regulate GFR. NOS1β is the primary splice variant and contributes to most of the NO generation by the macula densa. Recently, several studies from our laboratory demonstrated the decisive role of macula densa NOS1β-modulated TGF response in the long-term control of GFR, sodium excretion and blood pressure. However, whether the macula densa NOS1β-modulated TGF responsiveness plays a significant role in transition to CKD from AKI is unknown. In the present proposal, we propose to test our central hypothesis that following renal IRI, NOS1β expression and activity in the macula densa are decreased, which enhance TGF responsiveness and decrease GFR, thereby promoting transition to CKD. Rescue of macula densa NOS1 prevents AKI to CKD transition.