Old Dominion University

NIH Research Projects · FY 2023 · 2022-11

Project Summary Our preliminary work with low-income Black residents in Southeastern and Central Virginia suggests that preexisting mistrust in the public health establishment and other important government institutions has worsened during the pandemic, affecting decision making about following public health guidance related to COVID-19 testing and vaccination. In particular, participants in our current study report that a major barrier to COVID-19 testing is the expectation that a positive result will be used against them. This suggests that trust- building must be a cornerstone of any COVID-19 outreach efforts. The goal of this proposal is to address these concerns through the development of a community-led intervention co-developed with our partner Housing Collaborative Community Advisory Board (HC CAB). Our HC CAB partners report that our relationships with them helped overcome mistrust common in their communities. They described the way we interacted with them, rather than any specific content, led us to be viewed as trustworthy. Engagement with a strong relationship building focus is thus likely an important intervention in and of itself—in this study we will standardize a process of relationship and trust building to be used as core components of a community-led intervention promoting rapid COVID-19 testing. In Aim 1 we administer and evaluate a preliminary virtual Peer Mentor COVID-19 testing intervention developed with our HC CAB partners. It uses a Peer Mentor model in which HC CAB members guide other community members participating in the study through self-administering a rapid in-home test. The process will be virtual, with HC CAB member Peer Mentors and participants using digital access capacity provided and supported by the research team. In a second arm of the study participants will meet with research staff in group sessions meant to approximate the experience of being a member of a community advisory board. There will also be a control group which will only receive follow up assessment. In Aim 2 we use evaluation data to develop an adapted COVID-19 testing intervention. In Aim 3 we administer the adapted intervention.

NIH Research Projects · FY 2025 · 2022-09

Project Summary The critical challenge in the clinical management of newly-diagnosed localized prostate cancer remains distinguishing indolent from aggressive and life-threatening cancers. Biomarkers are urgently needed to identify those patients who harbor aggressive disease and will derive benefit from definitive treatment. We therefore, propose to apply complimentary proteogenomic-based discovery approaches to identify and then validate molecular features in prostate proximal fluids and tumor tissues that will be utilized in accurate early detection of aggressive forms of prostate cancer and improve disease risk stratification. The intended use of these biomarkers will be the early identification of men at risk for grade progression and improved risk- stratification for them. We have three biomarker development laboratory aims: 1) Validate our existing urine-based biomarkers for grade progression in a ProBE-compliant study selected from our own cohorts and the EDRN GU upgrading study. 2) Develop and validate urine and tissue-based biomarkers for the risk-stratification of MRI “invisible” high-grade lesions. 3) Develop and validate biomarkers to sub-stratify risk associated with deleterious germline BRCA2 variants. Our biomarker reference laboratory will develop and validate targeted clinically robust assays for multi-protein biomarkers panels. We will also develop decision algorithms that are cross-referenced for statistical rigor and benchmarked for optimal clinical performance. In addition to these BCC activities, we will develop robust PRM-MS assays and statistically rigorous decision tools for other EDRN BCCs and CVCs. Taken together, our EDRN biomarker characterization center will be a core part of the the EDRN ecosystem. We will continue to actively participate in trans-Network activities, and to share patient cohorts, protocols, datasets and analysis approaches and expertise. We will supplement these activities by focusing on promoting the growth of new and diverse talent in biomarker development through fostering junior investigator involvement across the full spectrum of biomarker development.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Social isolation is the quantifiable measure of social interactions and relationships, whereas loneliness is the perceived level of social isolation. These experiences are especially pronounced during life transitions. Social isolation and loneliness have deleterious effects on cardiovascular health. The development of a culturally sensitive, behavior change intervention that emphasizes the provision of social support, development of a sense of belonging, and foster feelings of social connectedness has the promise to reduce the growing public health epidemic of social isolation and loneliness while improving cardiovascular health. I have developed the proposed core training objectives to acquire a unique and valuable skill set in the following areas: 1) gain proficiency in community-engaged research (CEnR) for the development of effective physical activity (PA) interventions, 2) obtain knowledge and skills related to the health implications, evaluation, and treatment of social isolation and loneliness, 3) develop expertise in mixed methods study design to conduct behavioral randomized control trials (RCT), and 4) acquire knowledge of the health and culture of veterans. Secondary training objectives include professional development to increase the impact of manuscript submissions and presentations, develop persuasive grant writing skills, and conduct ethical and responsible research. The training aims will be accomplished through a combination of formal and informal coursework, directed readings and webinars, behavioral and social science research trainings related to CEnR, RCTs, PA interventions, veterans, and practical research experiences. The research objective is to develop and examine the feasibility and acceptability of a group-based PA intervention to reduce social isolation, loneliness, and blood pressure among an at-risk population going through a significant life transition, veterans reintegrating to civilian life. To achieve this objective, the proposed research aims include: 1) characterizing the needs related to PA, social isolation, loneliness, and social connectedness among veterans reintegrating to civilian life, 2) designing a group-based PA intervention supported by digital technologies using CEnR among this population, and 3) evaluating the feasibility and acceptability and preliminary health effects of the group-based PA intervention supported by digital technologies developed partly in Aim 2 among a sample of veterans reintegrating to civilian life. The environment at San Diego State University, along with my mentorship team, form a uniquely suited setting to expand my training and research skills in pursuit of my long-term career goal of becoming a successful independent investigator, conducting large-scale, community-based PA promotion RCTs.

NIH Research Projects · FY 2024 · 2022-08

Nanosecond pulsed electric field (nsPEF) is a new modality for neuromodulation, with unique capabilities qualitatively different from the conventional electrostimulation. The potential benefits of nsPEF include but are not limited to prolonged stimulation with little or no electrochemical side effects; excitation at lower thresholds; selectivity based on cell charging time constant; the capability of choosing between stimulation, inhibition, and ablation; and achieving these effects non-invasively, either for outpatient deep brain stimulation or for tumor ablation. The primary effect of nsPEF is a rapid build-up of cell membrane potential (MP). Real-time measurements of MP kinetics are a key to predicting the outcomes of nsPEF stimulation. They are also a key to understanding bipolar cancellation, a unique feature that enables interference targeting of nsPEF for non-invasive neuromodulation. However, membrane charging by nsPEF occurs on a nanosecond time scale, much faster than could be resolved by the existing electrophysiological and imaging methods. We have addressed this challenge by implementing strobe pulsed laser microscopy for MP imaging with better than 50 ns accuracy. In this one-of-a-kind set-up, cells loaded with a fast voltage-sensitive fluorescence dye are exposed to high-power momentary laser flashes (5 kW, 6 ns). The flashes are dynamically synchronized with nsPEF stimulation of target cells. Photos of fluorescence taken at different times during and after nsPEF show the real-time dynamics of MP changes and how these changes culminate in downstream effects, such as opening of voltage gated ion channels, initiation of action potentials, and nanoelectroporation. We will employ this all-new set-up for understanding fine mechanisms and principles how neurons respond to the nanosecond electric stress. We will characterize nsPEF parameters needed to evoke the desired neuromodulation effect and tune the interference targeting protocols to achieve this effect at a distance from stimulating electrodes. We will perform finite element modeling of the electric field thresholds and use our in vitro results to define the feasibility and nsPEF requirements for non-invasive deep brain stimulation. This project will generate new basic knowledge of neuronal function, including nanosecond-scale biophysics of the cell membrane and ion channels. We will systematically characterize nsPEF neuromodulation effects and link them to dielectric and physiological properties of neurons and to nsPEF stimulation parameters. This in vitro project will utilize R21 “high risk, high reward” concept to collect mechanistic and quantitative data necessary for animal and human studies of nsPEF neuromodulation.

NIH Research Projects · FY 2026 · 2022-02

Circulating tumor cells (CTCs) are exposed to various insults thought to reduce their survival including lack of anchorage and trophic support from the primary tumor microenvironment, immune-mediated destruction and exposure to hemodynamic forces that may mechanically destroy them. However, the relative contribution of these factors to CTC survival and their overall role in metastasis is unclear. It has recently been shown that cancer cells from many tissue origins actively resist destruction by fluid shear stress (FSS), implying that viable CTCs are not mechanically fragile as suspected. Our long-term goal is to understand the biomechanical influences on CTCs and how this contributes to metastasis. The objective of this proposal is to determine the mechanism of FSS resistance in cancer cells and its role in metastatic colonization. Our central hypothesis is that mechano-adaptation of viable CTCs to FSS promotes their survival in the circulation and “primes” them for subsequent events in metastasis. Our hypothesis is based on our previously published and preliminary data presented below as well as recently published data from others which is supportive of our hypothesis. The rationale for the proposed research is that once we understand the mechanism underlying FSS resistance; this would represent a novel therapeutic approach aimed at decreasing the survival of CTCs by enhancing their destruction due to the mechanical forces that naturally exist in the circulation. Guided by preliminary data, the central hypothesis will be tested by pursuing the following specific aims: 1) Define molecular mechanisms of FSS resistance; 2) Determine the effect of FSS exposure on metastatic colonization; 3) Determine the effectiveness of inhibiting FSS resistance as an anti-metastatic strategy. To accomplish these aims, we will employ a forward genetic screen to identify novel genes that mediate FSS resistance and determine their involvement with RhoA- actomyosin interactions. Short-term survival of CTCs will be assessed using a novel mouse model to measure entrapment of intact CTCs in the lung and destruction of CTCs measured by a plasma biomarker. We will validate novel genes identified in a forward genetic screen in similar assays. We will determine the involvement of RhoA- YAP activation by FSS in supporting the survival and extravasation of cancer cells lodged in the microcirculation. Finally, we will test the potential of clinically actionable drugs that sensitize cells to FSS as well as conditional RhoA/YAP knockdown to block productive metastatic colonization in mouse models. The proposed research is innovative because, it represents a paradigm shift from the idea that CTCs are mechanically fragile by elucidating a mechanism whereby viable CTCs actively resist destruction by hemodynamic forces and drives further events in metastasis. The proposed research is significant because defining the mechanisms and consequences of FSS resistance in CTCs will open entirely new diagnostic and therapeutic possibilities in cancer patients.

NIH Research Projects · FY 2025 · 2022-01

Project Summary Cardiomyopathies are the most common genetic cardiovascular disease worldwide. The presence of cardiac troponin variants accounts for at least 15% of all familial cardiomyopathy cases. Cardiac muscle contraction is regulated by free intracellular Ca2+ concentration via two thin filament regulatory proteins – tropomyosin and troponin complex. Ca2+ binding to troponin displaces tropomyosin from myosin-binding sites and allows formation of myosin cross-bridges, which on their own contribute to thin filament activation. Troponin complex is composed of Ca2+ sensing troponin C, actin-binding troponin I, and Tm-bound troponin T. For decades, the helical approach to electron microscopy reconstruction of the thin filament eliminated information on the structure of the Tn complex. Hence, the complex interactions among components of the thin filament remained unknown. We developed cryo-EM non-helical algorithm to the reconstruction of native cardiac thin filaments to reveal the structure of the whole troponin complex at physiological Ca2+ levels. We show that the thin filament is comprised of an array of Ca2+-free and Ca2+-bound non-equivalent troponin complexes with short-range cooperativity between adjacent units. Troponin variants associated with inherited cardiomyopathies affect thin filament Ca2+- dependent activation. We hypothesize that: (1) dilated (DCM) or hypertrophic (HCM) cardiomyopathy variants in troponin affect thin filament regulation by: (a) altering the equilibrium between Ca2+-free and Ca2+-bound troponin complexes via conformational changes in Ca2+-sensing troponin C unit; and/or (b) altering the distribution of Ca2+-free and Ca2+-bound troponin complexes by changing communication between the adjacent troponins along and across the thin filament. To test our hypothesis we chose 4 strategically located mutations. In Aim 1 we will utilize pathogenic variants in troponin C located in distal parts of Ca2+-sensing troponin N-lobe to evaluate how they affect the equilibrium between Ca2+-free and Ca2+-bound troponin complexes, and if they affect communication between troponin units that may curb activating effect of rigor myosin-S1. In Aim 2 we will focus on highly penetrant pathogenic variants in troponin T located in N-terminus of troponin T, which stabilizes the interaction between tropomyosin cables belonging to adjacent troponin units. We will evaluate how these mutations affect the communication between neighboring troponins and activation by myosin-S1. To reveal how distal parts of the troponin complex (Ca2+ sensing troponin C and N-terminus of troponin T) communicate, we will use a Ca2+ sensitizer, which binds to troponin C to revert effects of a troponin T variant. Our multidisciplinary, multi-PI approach with collective expertise in structural, functional and computational methods will reveal how the complex interactions between components of the thin filament make heartbeats possible. Successful execution of the aims may set the ground for the development of tailored therapies that could potentially modulate the structure of the thin filament to treat various forms of heart disease.

NIH Research Projects · FY 2021 · 2021-09

Project Summary Lack of proper vascularization leads to the ultimate failure in treatment of critical-sized craniomaxillofacial defects. The large size of the defect obstructs penetration of blood components from the surrounding environment into the inner parts of the defect, and thus hinders vascularity. In such situations, vascular endothelial growth factor (VEGF) is the most effective factor that can reestablish the oxygen supply to tissues. While applying external VEGF is a key means for blood vessel formation in critical-sized defects, its slight uncontrolled administration is risky and can be tumorigenic. Thus, conventional methods cannot be used for encapsulation and delivery of VEGF. In this proposal, we will develop a new on-chip method for delivery of VEGF with precise and sustained release capabilities using a microfluidic platform. Our novel design allows making monodispersed particles in a highly controllable and reproducible manner, providing us with the ability to fine- tune the size, microstructure, loading capacity and release rate of particles, in addition to balancing the pH and maintaining the VEGF bioactivity. Release of VEGF must not be only controlled and sustained, but also highly localized in the region of the defect as moving the VEGF-loaded particles into unwanted areas is not favorable and can be risky. Thus, in another strategy, the VEGF-loaded particles will be immobilized onto a new 3D-printed scaffold specifically designed for critical-sized defects. The design of this novel scaffold (filed for patent) is inspired by reinforced concrete, in which reinforcing Rebars are embedded in the host material to enhance the mechanical properties of the scaffold (100-375 times improvement). In other words, it is a hybrid scaffold, made of two components: 1) Skeleton Rebars: non-porous and slowly-biodegradable constituent undertaking mechanical necessities of the scaffold, and 2) Host Component: porous and rapidly-biodegradable constituent undertaking biological necessities of the scaffold. Although the mechanical strength of Rebars is the property that makes the scaffold appropriate for critical-sized defects, another functionality of the Rebar, which is its slow degradability (6 months), makes the design a perfect choice for the VEGF delivery purpose. Rebars will provide us with the opportunity to immobilize VEGF-loaded particles on a solid surface and not let the particles move elsewhere. The immobilization process itself is a new method developed in our lab that can firmly attach these particles to the rebars of the scaffolds. The VEGF-loaded scaffold will undergo a detailed in vitro analysis and release adjustment inside a bioreactor, which can mimic the body condition. The VEGF release profiles will be adjusted to reach the target value (1.2 ng/ml per day per cm3 of scaffold), and the comprehensive in vitro analyses will evaluate the osteogenesis and angiogenesis characters of the construct. The optimized VEGF-loaded scaffold will undergo a detailed in vivo study using critical-sized alveolar bone defects in New Zealand white rabbits. The new bone formation and angiogenesis will be fully studied to assess the functionality of the VEGF-loaded scaffold in comparison with a VEGF-free scaffold, as well as defects treated with a current therapeutic modality.

NIH Research Projects · FY 2025 · 2021-08

Abstract Although people living with HIV (PLWHIV) have comparable life-expectancy as the HIV-negative population does, their life-quality is still deeply compromised due to the prevalence of neuropsychiatric disorders including depression and anxiety. The main pathological changes in the brains of those people include aberrant microglial (Mg) activation and neuronal injuries. Drug abuse is a high comorbidity of HIV infection and abused drugs could exaggerate the existing neurologic complications in PLWHIV. The detailed mechanisms underlying such phenomenon remain much elusive. Several contributing factors have been suggested for such neurological disorders including the continued expression HIV proteins such as trans-activator of transcription (TAT), the long-term use of antiretrovirals (ARVs), and drugs of abuse. Our preliminary data demonstrated that :1) NLRP3 inflammasome signaling was involved in HIV-TAT-mediated Mg activation; 2) cocaine activated Mg through dysregulating miR-124/TLR4 axis, which could be reversed by inhibition of NLRP3 inflammasome; 3) combination ARVs used in clinical practice (tenofovir:TFV, emtricitabine:FTC, and dolutegravir:DTG) could activate Mg via lysosomal dysfunction; 4) co-exposure of Mg to three agents (TAT, cocaine, and ARVs) intriguingly resulted in increased activity of NLRP3 inflammasome in vitro; 5) IL1β, the final executor of NLRP3 inflammasome activation, decreased the spine density and increase the glutamate receptor ionotropic NMDA subunits (Grins) in vitro; and 6) increased NLRP3 inflammasome activity was shown in the brains of SIV-infected macaque. Based on these findings and two distinct steps of NLRP3 inflammasome activation, we hypothesize that exacerbated NLRP3 inflammasome activation in the context of HIV-TAT/HIV, cocaine, ARVs will lead to exaggerated Mg activation and neuronal injuries, which are responsible for the high incidence of neuropsychiatric disorders in PLWHIV with cocaine use. We will test this hypothesis in the following two specific aims (SA) using complimentary in vitro and in vivo approaches. SA1: Investigate the role of NLRP3 inflammasome in Mg activation and neuronal injuries in the context of HIV-TAT/HIV, cocaine, & ARVs. We will split this SA into three sub aims. SA1A will explore the detailed mechanisms responsible for exaggerated NLRP3 inflammasome activation in vitro; SA1B will explore the mechanisms underlying NLRP3 inflammasome-mediated neuronal injuries; and SA1C will investigate the status of NLRP3 inflammasome and neuronal injuries in archived SIV- infected macaque brains and HIV-infected individuas with or without cocaine use. SA2: Explore the potential therapeutic effects of NLRP3 inflammasome inhibition on neuropsychiatric behaviors in HIV iTat mice in vivo.

NIH Research Projects · FY 2024 · 2021-05

G-RISE at Old Dominion University Project Summary/Abstract Old Dominion University (ODU) is a research-active, urban-based university located in the Hampton Roads region of Southeastern Virginia. It enrolls 24,176 students (19,372 undergraduate and 4,804 graduate students) with approximately 33% from underrepresented minority (URM) groups and 56% females. This project will implement the NIH/NIGMS Graduate Research Training Initiative for Student Enhancement (G-RISE) program at ODU to support graduate research training of 20 NIH-supported and 4 ODU-supported Ph.D. students over a five-year grant period. The overarching goal is to prepare underrepresented (UR) groups to enter and succeed in the biomedical research workforce. The objectives are to recruit, retain, and train a diverse pool of UR students in fields of biomedical research through research experiences, mentoring, and educational and career development activities. The expected outcomes are 80% trainee 5-year time-to-degree and 100% placement into post-doctoral research studies and/or employment in biomedical research related occupations. The G-RISE at ODU program will be built on existing and successful UR undergraduate education programs at ODU. It will maximize access to research careers in biomedical research for UR students through ODU partnerships with the Virginia Bio consortium (+ 270 bioscience firms) and Brookhaven National Laboratory. The program will commence with a structured and intensive mandatory six-week summer doctoral bridge and will continue into the academic years with academically challenging coursework, year around research experiences, intellectual development, mentorship, and advising. In addition to scholarship support, the program will provide career development activities and strong programmatic values to create an environment that promotes and nurtures UR student success. The designated PI/PD of the G-RISE at ODU program is a tenured URM female faculty member in Electrical Engineering and the Executive Director of the ODU Frank Reidy Research Center for Bioelectrics. She has trained over 60 students (59% African American, 35% women), one of whom is a Rhodes Scholar (African American female). The designated Co-PI/PD is a tenured URM male faculty member in the Department of Chemistry & Biochemistry and the PI/PD of the current NIH MARC program at ODU. He has trained over 120 students (92% African American, 90% women). They will guide the G-RISE at ODU program design as well as serve as role models for the UR doctoral students.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT The overarching goal of my proposal is to acquire technical and professional skills to become an independent investigator at a leading academic institution and develop a research program deciphering the molecular mechanism of inflammation induced prostatic tissue remodeling and fibrosis. This will be pursued through a scientific project that will determine whether osteopontin, a pro-fibrotic secreted phosphoprotein, stimulates prostatic inflammation, fibrosis, and lower urinary tract dysfunction. My training is focused on four key areas: 1) functional testing of mouse urinary function, 2) developing biomedical engineering technologies to study prostatic fibrosis in vitro, 3) testing and further developing animal models to study inflammation-induced prostatic fibrosis and its consequences on urinary function and, 4) gaining essential training in immune-regulated tissue remodeling. UW-Madison and the UW O`Brien Center for Benign Urology Research presents a unique environment for the proposed research and career development activities. This includes seminars presented by local and national leaders of the field, career development activities of several institutes across campus, clinical training of the Department of Urology, and specific training in immunopathology and tissue engineering. Lower urinary tract symptoms (LUTS) secondary to benign prostatic diseases deteriorate the quality of life as men age. The treatment of male LUTS costs $4 billion annually and presents an economic burden on our healthcare system. It has been recently identified that prostatic inflammation and fibrosis are associated with LUTS, but the exact contribution of these mechanism to urinary dysfunction is unknown. Medical therapies targeting inflammation and fibrosis could enhance drug development and provide novel molecular targets for LUTS. Based on my preliminary studies, I hypothesize that inflammation-induced osteopontin levels stimulate prostatic fibrosis and lower urinary tract dysfunction (LUTD). The hypothesis will be tested by the following aims: 1) Test the hypothesis that OPN is required for inflammation-induced prostatic collagen accumulation and LUTD, 2) Test the hypothesis that OPN induces prostatic fibrosis. The proposal will provide novel detection of collagen deposition, 3D in vitro and in vivo models of prostatic fibrosis. It will also decipher the specific role of inflammation-induced prostatic fibrosis in lower urinary tract dysfunction. This will be achieved by capitalizing on recently established prostatic inflammation models and state-of-the-art urinary physiological tests uniquely available at the UW-Madison.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Smoke-free housing (SFH) has recently been mandated by the U.S. Department of Housing and Urban Development (HUD) to protect public housing residents from secondhand smoke (SHS) exposure. However, our preliminary work suggests that SFH might not be effective. In focus groups, smokers in low-income housing have reported to us that they are primarily motivated by fear of punishment, rather than an obligation to comply with SFH, which they perceive to be unfair. They describe a situation in which many smokers respond by hiding their smoking inside their apartments—where they think they will be less likely to be seen—when compliance with SFH is perceived to be an inconvenience or even unsafe (e.g., if they would have to walk across their community at night to smoke off the property). This suggests that the perceived legitimacy of SFH is low—residents feel so strongly about its unfairness that they are unlikely to comply with it on their own. This means that efforts to increase resident compliance by relying on threats of punishment could be counterproductive. Residents also report several barriers to compliance with SFH that they also perceive to be unfair (e.g., banning smoking in public housing but allowing it on other types of properties). These barriers vary between housing authorities and represent important differences in SFH implementation strategy. Principles and approaches from community-based participatory research guide this study. In Aim 1 we assess factors associated with perceived legitimacy and compliance across six housing authorities. We will use a mixed methods approach to establish: (1) who exhibits lower perceived legitimacy of SFH, and under what circumstances, and (2) what property and organizational factors that differ between and within housing authorities could affect SFH implementation and resident compliance. In Aim 2 we test associations between perceived legitimacy, SFH implementation strategy, and several markers for SHS (fine particulate matter, airborne nicotine, and exhaled CO). We hypothesize that: (a) perceived legitimacy will be related to differences in SFH implementation, (b) differences in SFH implementation strategy will affect SFH compliance, as measured by SHS, and (c) low perceived legitimacy of SFH will mediate the impact of implementation strategy on SFH compliance. In Aim 3 we develop a scalable implementation strategy for SFH that to improve resident compliance and perceived legitimacy. Community advisory boards will be used to inform this process to ensure that solutions are grounded in real-world experiences.

NIH Research Projects · FY 2023 · 2020-08

SUMMARY/ABSTRACT Beta cell failure, microvascular endothelial cell dysfunction, fibrosis, and calcification are clinically significant problems in type 2 diabetic (T2D) patients because they cause myocardial infarction, stroke, peripheral artery disease, retinopathy, nephropathy, cardiomyopathy, and wound healing delay. However, the detrimental mechanisms responsible for these pathologies in T2D are not completely understood. Current therapies for T2D neither halt nor reverse beta cell failure, endothelial cell dysfunction, nor the tissue complications. Therefore, there is a critical need for the identification of mechanism-based, treatable targets to improve beta cell and microvascular function, to reduce fibrosis and calcification, and to limit the abnormalities of multiple tissues and organs in T2D. Cytokine and adipokine secretion is increased in T2D, which affects beta cell and microvascular function and structure. Specifically, the level of the pro- inflammatory cytokine interleukin-12 (IL-12) is increased in adolescent and adult T2D patients. However, it is unknown whether the inhibition of IL-12 protects beta cell and microvascular function, thereby reducing fibrosis and calcification in multiple organs. Furthermore, the mechanism by which increased IL-12 might cause these pathologies is unknown. The premise is that IL-12 administration to non-obese mice leads to diabetes, liver toxicity and fibrosis, kidney damage, and atherosclerosis, supporting a detrimental role of IL- 12 in diabetes, and multiple tissues and organs damage. We hypothesize that increased IL-12 in T2D mice causes beta cell dysfunction, hyperglycemia, insulin resistance, microvascular endothelial cell dysfunction, fibrosis, and calcification through inflammation, endoplasmic reticulum (ER) stress, and autophagy mechanisms. To test the hypothesis, we proposed the following aims: Aim #1: IL-12 causes beta cell and endothelial cell dysfunction, fibrosis, and calcification in T2D. We will assess whether IL-12-induced pathology in T2D can be abrogated using genetic deletion of IL-12 or neutralizing IL- 12 antibody; Aim #2: IL-12 induces pancreatic islet inflammation in T2D. We will examine if genetic deletion of IL-12 or neutralizing IL-12 antibody in T2D mice attenuates the inflammation in pancreatic islets, and subsequently improves beta cell and endothelial cell function, and reduces fibrosis and calcification; Aim #3: IL-12 induced beta and endothelial cell dysfunction, fibrosis, and calcification are dictated by an ER stress mechanism in T2D. We will illustrate the mechanisms in beta cells and endothelial cells whereby IL-12 leads to beta cell and endothelial cell dysfunction, fibrosis and calcification in T2D; Aim #4: IL-12 induced beta cell and endothelial cell dysfunction, fibrosis, and calcification are driven by an autophagy mechanism in T2D. We will elucidate the mechanisms in beta cells and endothelial cells whereby IL-12 leads to beta cell and endothelial cell dysfunction, fibrosis, and calcification in T2D.

NIH Research Projects · FY 2023 · 2020-07

ABSTRACT — The central mechanisms involved in hypertension-induced vascular pathology, a public health crisis, remain unknown. There is still a significant rate of adverse events in hypertensive patients prescribed these therapeutic and 2/3 of hypertensive patients are still resistant to these therapies. Thus, the critical unmet need is to identify mechanism based-treatable targets to rescue vascular function and structure in established hypertension. The pilot data showed that transferring healthy Treg into a mouse with established hypertension-induced by angiotensin II (Ang II) infusion improved vascular endothelial function and structure. We showed an increase in stromal interaction molecule 1 (STIM1) expression in Treg that could be responsible for Treg apoptosis by Nox2 and endoplasmic reticulum (ER) stress- dependent mechanisms. The overexpression of STIM1 in Treg cell caused Treg cell apoptosis. The depletion of dendritic cells in hypertensive mice improved arterial function and reduced arterial fibrosis and calcification through a reduction in INFγ and IL-1β release from dendritic cells and the inhibition of the ER stress in the endothelial cells. The central hypothesis is that STIM1 overexpression in Treg cells, through ROS and ER stress mechanism, cause Treg cells apoptosis and decrease IL-10 release, which increases dendritic cell activity leading to an increase in pro-inflammatory cytokines release (INFγ and IL-1β) and a decrease in anti-inflammatory IL-10 release causing the induction of the ER stress in endothelial cells and vascular pathology. To advance the Translational Sciences, we will test the hypothesis in two-kidney one- clip (2K1C) hypertensive mice Ang II-dependent. Specific Aim #1: To determine that in established hypertension, STIM1 expression is increased in Treg cells causing Treg cells apoptosis, a decrease in IL-10 release, and vascular pathology. Thus disrupting STIM1 expression in Treg cells would restore Treg cells number, IL-10 levels, and improve vascular endothelial function and reduce fibrosis and calcification in established hypertension. Specific Aim #2: To delineate that the decrease in IL-10 release, because of apoptosis in Treg, increases dendritic cells activity to release IFNγ and IL-1β and blunt IL-10 release, which causes vascular pathology via the induction of the ER stress mechanism in endothelial cells, and therefore depleting dendritic cells or manipulating the ER stress in endothelial cell improve vascular endothelial function and reduce fibrosis and calcification in established hypertension-induced by 2K1C. These studies are central to the mission of the NHLBI and address all Goals and multiple Strategies outlined in the NHLBI Strategic Plan. These studies will address 1) a need to further illuminate the biological mechanisms and pathological processes of the contribution of the immune cells, 2) The interaction between the immune system and the vascular system as a priority research topic and, 3) To advance the Translational Sciences, we will test the hypothesis in 2K1C mice model.

NIH Research Projects · FY 2024 · 2019-08

PROJECT SUMMARY/ABSTRACT The primary objective of this proposal is to facilitate the development of Dr. Brachova into an independent research scientist. This objective will be accomplished through a combination of active mentorship, didactic training, enrichment activities, and research. The mentorship phase is well described by her mentors and includes weekly contact and formal course work. The core of the research focuses on the role of a post- transcriptional regulation mechanism known as RNA editing in overall oocyte quality. Defects in oocyte growth and meiotic maturation represent a significant cause of human birth defects, miscarriage, and infertility. The contribution of RNA post-transcriptional modifications is emerging as an important regulator of RNA stability and oocyte quality, however the role of adenosine deaminase acting on double stranded RNA (ADAR1), which catalyzes the deamination of an adenosine (A) into inosine (I) remains unexplored. The goal of this research is to determine the effect of ADAR1 RNA editing on RNA stability and oocyte competence in young and old mice. Preliminary data show that ADARFL/FLZP3-Cre oocytes have reduced embryonic developmental potential, suggesting that ADAR1 is a novel factor that contributes to female fertility. We also show that oocytes and eggs from aged individuals have reduced RNA editing efficiency, suggesting that RNA editing deficiencies could be involved in female age-associated decline of fertility. The central hypothesis is that ADAR1 RNA editing is a novel mechanism for maintaining female fertility through the regulation of RNA stability during oocyte growth and early embryonic development. The hypothesis will be tested in two independent specific aims. Specific Aim 1 will assess the role of ADAR1 A-to-I RNA editing in the regulation of maternal mRNA dosage in the oocyte and early embryo. Specific Aim 2 will determine how ADAR1 A-to-I RNA editing effects mRNA degradation in oocytes. The proposed research will utilize oocytes and eggs from young and maternally aged mice to explore the role of A-to-I editing in oogenesis and age-related reduction in fertility. This proposal represents an innovative approach to study oocyte competence, and has the potential to expand our understanding of female fertility. Furthermore, the studies proposed herein are integral to enhance the scientific training of Dr. Brachova and enable her to accomplish her goal of achieving independence and becoming an independent scientist in the field of female reproduction.

NIH Research Projects · FY 2023 · 2019-02

PROJECT SUMMARY/ABSTRACT There is a need to develop next-generation multipurpose prevention technologies (MPTs) that provide long-acting (LA), simultaneous systemic delivery of products for the prevention of HIV acquisition and unintended pregnancy. We propose to develop a novel silica hydrogel-based LA injectable depot system delivering dolutegravir (DTG), a proven and potent HIV integrase strand-transfer inhibitor (INSTI), and levonorgestrel (LNG), a licensed contraceptive agent. In the first (R61) phase of the project (Specific Aims 1-3), the main objective is to achieve milestones demonstrating feasibility of developing a particle-based hydrogel depot system capable of providing at least 3 months duration for both drugs, shorter tail pharmacokinetic (PK) profiles, and no drug-drug interaction (DDI). In Specific Aim 1, we will conduct iterative formulation development to produce and screen prototype combinations for initial feasibility, which will then be evaluated preclinically in Specific Aim 2 in rat and non-human primate models to determine optimal PK/pharmacodynamics (PD) profiles and characterize safety and DDI, to support selection of a lead MPT formulation. In Specific Aim 3, we will engage with potential end-users in a US region with high HIV incidence to gain a more in-depth understanding of user preferences of product attributes. In the second (R33) phase (Specific Aims 4-6), the main objectives will be to expand product development efforts to characterize the lead formulation and obtain IND-enabling feedback from the FDA in a pre-IND meeting, conduct POC contraceptive efficacy and HIV prophylactic efficacy in animal studies, and define the optimal dosing regimen and target product profile (TPP) based on end-user input. In Specific Aim 4, formulation optimization activities will be performed to improve the harmonized PK profile and duration for each drug and to better meet the product attribute preferences prioritized by end users. Preclinical proof-of-concept data will be generated using a rat contraceptive efficacy model and the well-established repeated, low dose SHIV challenge models using pigtail (intravaginal) and rhesus (intrarectal) macaques at CDC. Upon identification of the lead formulation, we will also draft a toxicology testing plan, clinical study design and clinical development plan to support a pre-IND meeting with the FDA (Specific Aim 5). Finally, in Specific Aim 6, we will build upon the formative work of SA3 with a discrete choice experiment to understand user and provider preferred product attributes for the LA MPT injectable, in order to refine the TPP and guide on-going product development efforts. In summary, this project proposes to develop, through preclinical proof-of-concept, a LA MPT injectable with a strong regulatory path for future clinical advancement, providing a significant advancement of a next-generation HIV prevention and contraceptive product that may fit into the lifestyles of at-risk women most in need.

NIH Research Projects · FY 2025 · 2016-06

Project Summary/Abstract The overall prognosis for Glioblastoma Multiforme (GBM) brain tumors is extremely poor with a median survival of about one year from diagnosis and five-year survival rates of only 6.9%. Multiple biological characteristics contribute to the lethality of GBM including rapid, uncontrolled proliferation throughout the restricted cranial space supported by their pro-angiogenic nature and ability to rapidly develop therapeutic resistance resulting in tumor recurrence (TR). Identification of early TR is complicated by chemoradiation induced toxicity which can present initially as pseudo-progression and later as radiation necrosis (RN) condition that occurs in 25% of all GBM. Currently, invasive stereotactic brain biopsy at the site of suspicion remains the only resort for TR confirmation. This proposed renewal study will develop novel computational models and AI methods for analyzing brain TR, radiation necrosis (RN) and postoperative rapid early progression (REP); and to rigorously validate the models using both in-house and public domain patient data in collaboration with the ReSPOND consortium. This goal will be accomplished via the following aims: (1) Multi-center multimodal patient data collection, data harmonization, and robust TR, edema and RN volume segmentation and tracking; (2) Co-analysis of imaging and molecular features for TR and progression analysis; (3) Explainable AI modeling with uncertainty analysis to understand TR and progression; and (4) Evaluation and cross-validation of the proposed methods. The proposed co-analysis of radiomics, proteomics, and histopathology data is expected to enable robust TR, progression and REP analysis that may be critical to stratification of aggressive versus non-aggressive brain tumors for patients, with the potential for targeted therapy transcending the current limitations of the one-size- fits-all radiation treatment planning paradigm. In our parent R01 project, we made considerable progress in: (i) addressing critical barriers related to brain tumor volume segmentation, tumor growth tracking, glioma diagnosis and grading; (ii) GBM patient survival prediction using multi-modal radiology, molecular and histopathology patient data; (iii) molecular prediction of diffuse low-grade glioma using advanced radiomics features; and (iv) prediction of low-grade glioma progression using radiomics features. Recently, we showed the feasibility of multiresolution radiomics texture features to discriminate TR from RN. Very recently, our pilot study showed the feasibility of quantitative co-analysis of radiomics, proteomic and clinical patient data for REP and patient survivability prediction. Furthermore, we have been consistently ranked among the top teams in Global Challenges on Brain Tumor Segmentation (BRaTS), Brain Ischemic Segmentation, and Patient Survivability Prediction since 2013. These advances will be foundational in achieving the aims of this proposed study.

NIH Research Projects · FY 2024 · 2015-09

Project Summary E-cadherin is the primary mediator of strong cell-cell adhesion between epithelial cells and plays an essential role in the morphogenesis and maintenance of epithelial tissues. E-cadherin is also a known mechanosensor that actively responds to the levels of inter-cellular forces and resides in a microenvironment formed by adjoining epithelial cells. The long-term goal of the project is to understand how the mechanical regulation of E-cadherin adhesion leads to a cohesive yet dynamic multi-cellular architecture in epithelial tissues. The goal of the proposed project is to delineate the mechanism by which forces are transmitted via E-cadherin adhesions and to uncover how epithelial cells sense cell-like stiffness laterally via E-cadherin adhesions. The E-cadherin-β-catenin-α-catenin complex directly and indirectly couples to actin to transmit cell-generated forces. Firstly, while the α-catenin-vinculin link, which plays a role in force sensing is thought to be the primary force transmission pathway, we recently found that, surprisingly, α-catenin is not essential for force transmission. Therefore, we will test the hypothesis that the less studied β-catenin-vinculin link is an alternate significant mechanism of force transmission at E-cadherin adhesions. We will test this by using mutant versions of vinculin and α-catenin deficient in binding β-catenin and vinculin, respectively, and corresponding knockout cell lines. We will use traction force microscopy with E-cadherin-coated soft substrates to avoid the confounding factor of vinculin’s mechanical role in cell-matrix contacts. We will also use magnetic pulling cytometry with E-cadherin-coated beads and biaxial stretching of cell islands to assess the adhesion strength at multiple scales. Secondly, while E-cadherin has been shown to sense the stiffness of E-cadherin-coated soft substrates, it is still unclear if epithelial cells sense cell-like stiffness laterally via E-cadherin adhesions. To test this, we will devise a biomimetic model of E- cadherin in a physiologically relevant geometry, in which cells interface laterally with an E-cadherin-coated, stiffness tunable, soft surface. We will test the hypothesis that lateral sensing of cell-like stiffness modulates E-cadherin density, cell dynamics, Rho and YAP levels. We will also test whether this lateral stiffness sensing is dependent on the α-catenin-vinculin mechanotransduction axis. We will use biomimetic soft substrate fabrication, mutant version of α-catenin deficient in binding vinculin in α-catenin knockout cells, sensors/indicators of Rho and YAP, and perform live-cell imaging and immunofluorescence to accomplish this. Knowledge gained on cell-to-cell force transmission and epithelial cell sensing of neighboring cell mechanical properties will be crucial in understanding the context-dependent biophysical control of E- cadherin adhesion. This will be essential to better understand the functional basis of the role of E-cadherin in mediating epithelial tissue integrity, mechanical coherence and its dysregulation in disease states like cancer.