University Of South Alabama

NSF Awards · FY 2024 · 2024-10

This project aims to serve the national interest by transforming STEM faculty understanding of effective instructional practices, which could ultimately improve student success in undergraduate STEM education. The project aims to enhance STEM faculty engagement with the STEM education research literature and effective STEM teaching practices through an intensive summer workshop and an ongoing regional STEM faculty learning community. Project activities aim to engage STEM faculty from the University of South Alabama and their STEM instructor colleagues at Bishop State, Coastal Alabama, and Mississippi Gulf Coast community colleges. Project efforts are intended to build capacity across these campuses for future efforts to improve undergraduate STEM education. This project plans to create and sustain Faculty Learning Communities (FLCs) across multiple institutions in the Gulf Coast region to help develop a shared vision for improving equitable STEM education across three local community colleges and the University of South Alabama. An additional goal of the project is to increase STEM faculty’s knowledge of research about Evidence-Based Instructional Practices, which could improve STEM teaching and student success in STEM courses. The project aims to engage up to 30 faculty participants across the four institutions in one of three discipline-based FLCs and a week-long summer institute. The FLCs will be tasked with creating an annotated bibliography, built as a searchable database, that may be shared with other STEM faculty across the participating institutions and STEM departments for broader impact. The outcomes of the project will be gauged by assessment of changes in STEM Faculty participants knowledge of and comfort with STEM education research literature. Further, the project aims to build capacity for future undergraduate STEM education reform efforts and development of grant proposals. This project has the potential to have a lasting impact on STEM teaching and learning in the Gulf Coast region and contribute to greater understanding of how STEM faculty across community colleges and a 4-year institution can increase their engagement with evidence-based STEM teaching practices. The NSF IUSE: EDU Program supports research and development projects to improve the effectiveness of STEM education for all students. Through its Institutional and Community Transformation track, the program supports efforts to transform and improve STEM education across institutions of higher education and disciplinary communities. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-10

Carbohydrate Memristor Empowered Environmentally Sustainable Processor-in-Memory Nontechnical description: Artificial intelligence (AI) systems have profound influence on societal wellbeing of humans and fueling significant economic growth. However, operation of conventional computing architecture in AI systems as well as manufacturing and disposal of conventional computing devices lead to significant energy consumption, depletion of nonrenewable natural resources, and ecological deterioration. Therefore, a serious concern of environmental sustainability to such increasingly pervasive computing systems has been raised, and improvements in system performance, energy efficiency, and ecological friendliness require new devices and systems. In this project, a new environmentally-sustainable processor-in-memory system is proposed to benefit the entire computing community including mobile and wearable computing, cloud computing and data center, electronic sensing and controlling, communication and networking. This project will also contribute to the development of high-quality workforce skilled in design, fabrication, testing, and modeling of memory devices and processor-in-memory computing systems for the growing needs in the US. The students and postdoctoral scholar participating in this project will receive unique training in engineering problem solving and technology development, and their research and educational experience will be enhanced by complementary expertise and close collaboration between the two research groups. Technical description: Processor-in-memory systems implemented with memristors have great potential to perform complex AI computations faster and on a smaller footprint. The goal of this project is to address the environmental sustainability challenge in computing by developing a novel brain-inspired processor-in-memory system empowered by memristors made from carbohydrate materials for energy-efficient operation, renewable material resource, sustainable device manufacturing, and ecologically-friendly disposal. The carbohydrate materials will be naturally extracted from plants, vegetable, and fruits with low cost and waste generation. Innovative fabrication techniques for carbohydrate-based memristor and processor-in-memory system will be developed with reduced water use and chemical waste, greenhouse gas emission, and manufacturing related energy consumption. The processor-in-memory system will be constructed by implementing carbohydrate memristors and reconfigurable peripheral circuits to achieve sustainable computing, enable performance improvement, and ensure high operation efficiency, longevity, and reliability. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-10

This three-year REU Site: Interdisciplinary Research to Address Microplastics in Gulf Coast Region is hosted by the University of South Alabama. The project takes a multidisciplinary approach to exploring the impact of microplastics in the Gulf Coast region. Microplastics, which are tiny plastic particles less than 5 millimeters in size, have become a significant environmental concern, prevalent in oceans, rivers, and coastal areas. These pollutants not only harm marine life but also pose risks to human health and disrupt ecosystems. Microplastics are a very real issue with major economic impacts, especially with respect to Gulf Coast based tourism. Ten students each year will be recruited to engage in place-based research during the ten-week summer program. REU students can select projects featuring civil, coastal, and environmental engineering, mechanical engineering, computer and electronic engineering, and marine science. The research initiatives include the impact and degradation of plastics into microplastics, the detection and measurement techniques for microplastics, filters to mitigate microplastics, microplastics monitoring system, and repurposing collected microplastics for alternative applications. REU students will have the opportunity to work together as a team, conducting both fundamental research and hands-on experiments as they investigate real-world issues. Students will also receive formal training in ethics and codes of conduct, experimentation, data collection, systematic and critical thinking, writing skills, and team building. Participants will engage in research about microplastics pollution and developing effective solutions to protect marine ecosystems, promote sustainable practices, and enhance the long-term health of coastal regions. This three-year REU Site: Interdisciplinary Research to Address Microplastics in Gulf Coast Region is hosted by the University of South Alabama. The project takes a multidisciplinary approach to exploring the impact of microplastics in the Gulf Coast region. The primary goal for students is to engage in intensive research towards detecting, measuring, and remediating microplastics in coastal regions, while also understanding the impacts of sustainable practices. REU participants will have the opportunity to work together as a team, conducting both fundamental research and hands-on experiments as they investigate real-world issues. Student activities encompass five key areas: understanding impact and degradation of plastics into microplastics; developing detection and measurement techniques for microplastics; creating filters to mitigate microplastics; establishing a microplastics monitoring system; and repurposing collected microplastics for alternative applications. Through these initiatives, students will collaborate on fundamental research and practical experiments. Students will also receive formal training in ethics and codes of conduct, experimentation, data collection, systematic and critical thinking, writing skills, and team building. This project is jointly funded by the EEC REU and the Established Program to Stimulate Competitive Research (EPSCOR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-09

ABSTRACT Endothelial cell (EC) dysfunction initiates the development of hypertension (HTN), but the mechanisms are not fully elucidated. Given that HBP is the leading cause of cardiovascular diseases, it is critical to identify new opportunities to restore EC function in HTN. In a series of supportive preliminary studies, we identified progranulin (PGRN), an anti-inflammatory protein, as a novel regulator of EC function and BP. We recently published that PGRN deficiency induces vascular dysfunction and HTN, whereas treatment with recombinant PGRN (rPGRN) restores these cardiovascular outcomes via EphrinA2 and nitric oxide (NO) production. In this proposal, we are extending our knowledge on PGRN and BP and identifying endothelial PGRN as a new regulator of endothelial function and BP. In preliminary study, we show that Angiotensin II- induced HTN selectively reduces PGRN in mesenteric EC, whereas mouse with a selective deletion of PGRN in EC (PGRNEC Cre+) displays endothelial dysfunction, reduced circulating PGRN, and are more susceptible to develop HTN. In further preliminary studies, we identified a new function of angiotensin converting enzyme (ACE), which is to degrade endothelial PGRN and limits its vasodilatory function. These data suggest that reduced endothelial PGRN, driven by ACE-mediate cleavage, is a trigger for EC dysfunction and HTN. Mechanistically, we found that mesentery EC from PGRNEC Cre+ present suppressed AMP-activated protein kinase (AMPK), while overexpressing PGRN in mesenteric EC resulted in exacerbated PGRN secretion followed by AMPK activation and NO formation, which were blunted by blocking EphrinA2, suggesting that endothelial PGRN regulates AMPK activation and NO formation via EphrinA2 in an autocrine-dependent manner. In further preliminary studies, rescuing PGRN expression in mesenteric arteries ex vivo with rPGRN restored the endothelial function in PGRNEC Cre+, but it failed to produce vasodilation in our novel double knockout mouse – global PGRN deficient mice with lack of endothelial AMPK. We also demonstrated that AMPK is in oxidized form in EC from PGRN deficient mice. Finally, Nox1-derived ROS are elevated in EC from PGRN EC Cre+, and that pharmacological inhibition of Nox1 rescues the endothelial function in PGRN EC Cre+. These findings indicate that endothelial PGRN maintains EC function and BP by restricting AMPK oxidation and Nox1 activity. These novel findings inform the central hypothesis of this proposal: Endothelial PGRN, via an autocrine mechanism, regulates endothelial function and BP and in HTN ACE degrades endothelial PGRN affecting its bioactivity and contributing to cardiovascular outcomes. This hypothesis will be tested in the following aims: AIM 1: Determine if reduced endothelial PGRN levels facilitate the genesis and progression of HTN via Nox1- derived ROS, AMPK oxidation, and EC dysfunction. AIM 2: Determine if ACE cleaves endothelial PGRN and contributes to endothelial dysfunction and HTN.

NSF Awards · FY 2024 · 2024-09

This project will provide a path for ten Predominately White Institutions (PWIs) or new Minority Serving Institutions (MSIs) to evaluate, identify and change the policies, processes, and everyday practices that contribute to racial inequities in STEM education. Over nearly two years, these cohort institutions will be guided by experts through evidence-based and theory-informed strategies to reconcile explicit and implicit instances of systemic inequities that exist in their structures, cultures, policies, and practices. This process will significantly impact the experiences and outcomes of students, faculty, and administrators at MSIs, a large and growing sector of institutions whose work is reducing racial equity gaps in STEM degree completion nationally. Products from this project will also broadly benefit PWIs and be immediately useful for campuses that are already or soon to be engaged in equity-centered transformation efforts. The project includes a comprehensive dissemination strategy to create a forum for institutions and stakeholders with similar commitments to discuss, dissect, and advance this approach while adopting and adapting it for their college and university STEM programs. With an acute focus on improving racial equity among the STEM disciplines, the aim of the Determining Equity Readiness in Higher Education (DERHE): Empowering Student Success in STEM Education project is to develop and test a practical, comprehensive, and evidence-based strategy to identify and cultivate the readiness of IHEs to address systemic inequities within a STEM education context. The following five objectives, informed by racial equity and organizational change scholarship, will help achieve this goal: 1. Engage and enhance campus stakeholders' (faculty, staff, administrators) perspectives on systemic racism and the organizational and institutional factors that mitigate and impede students' achievements in STEM education. 2. Develop, test, and administer a survey of institutional readiness for equity-centered change to assess campus capacities related to addressing systemic racism in STEM education. 3. Use process mapping to evaluate existing organizational, structural, and cultural elements that contribute to racial inequities while providing the strategies to address and re-evaluate these practices to ensure equitable experiences and outcomes across racial groups. 4. Convene stakeholders, educators, policymakers, and practitioners to share promising practices, solicit feedback, and provide recommendations based on emerging and ongoing research findings. 5. Cultivate institutional capacity for increased efficacy, effectiveness, and scalability, as well as capacity-building strategies to ensure that the valuable insights from this project can be implemented by other institutions seeking to promote racial equity in STEM education. This project is funded through the Racial Equity in STEM Education activity (EDU Racial Equity). The activity supports research and practice projects that investigate how considerations of racial equity factor into the improvement of science, technology, engineering, and mathematics (STEM) education and workforce. Awarded projects seek to center the voices, knowledge, and experiences of the individuals, communities, and institutions most impacted by systemic inequities within the STEM enterprise. This activity aligns with NSF’s core value of supporting outstanding researchers and innovative thinkers from across the Nation's diversity of demographic groups, regions, and types of organizations. Programs across EDU contribute funds to the Racial Equity activity in recognition of the alignment of its projects with the collective research and development thrusts of the four divisions of the directorate. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-09

An award is made to the University of South Alabama to acquire a dual inverted selective plane illumination microscopy (diSPIM) light sheet system. The diSPIM light sheet system enables rapid acquisition of extended time lapse image data over a range of relevant biological volumes, from 0.03 to more than 300 µm3. The acquisition speed of light sheet systems is orders of magnitude faster than other high-end microscopes, while inducing significantly less photodamage. This allows simultaneous measurement of intracellular signals as well as the colocalization and distributions of proteins at subcellular resolution throughout large scale model systems. The diSPIM light sheet system is a scientific nexus for integrating concepts from engineering biological sciences, mathematics, and computer science. Broader training impacts include fostering multidisciplinary expertise that promotes a collaborative scientific training environments for the next generation of biological scientists, chemists, and engineers. Broader educational impacts include incorporation of advanced microscopy and image analysis capabilities in laboratory-based coursework and research opportunities. Broader outreach impacts include summer training experiences for high school and undergraduate students, providing access to cutting edge imaging and image analysis capabilities through summer research programs. This diSPIM system enables projects that support understanding and application of basic biological, chemical, and engineering principles over large length scales, with applications in marine biosystems, environmental toxicology, biochemistry, cell biology, and physiology. This award supports a broad array of projects at the intersections of biological sciences, environmental toxicology, engineering, and computer sciences. Project investigators explore fundamental mechanisms underlying the mechanics of vascular tissue, plant and animal development, movement, cellular communication, information transfer within biological systems, and the roles of rare populations of immune cells. The diSPIM light sheet system allows investigators to expand scientific inquiry over a broad range of length and time scales. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

This project, Magnetic Monopole Search with NOvA and Undergraduate Particle Physics Education with Fermilab and CERN at the University of South Alabama, funds the NOvA group at the University of South Alabama. A leading experiment on the intensity frontier, NOvA offers a rich source of data to both advanced researchers and beginning researchers, including undergraduate students. As part of the NOvA Exotics Group, this group will continue the search for fundamental particles with a special emphasis on slow magnetic monopole searches. In addition to this basic research, this project will give undergraduate students the opportunity to experience the international world of particle physics. Students will participate in a one-year program where they will be instructed in particle physics techniques and research, participate in the already successful study abroad program of European particle detectors (e.g. CERN), and travel to Fermilab to learn about the domestic particle physics program. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Sepsis, a dysregulated host response to infection with high morbidity and mortality, is characterized by a systemic inflammatory response and widespread vascular hyperpermeability leading to edema, organ dysfunction, and death. Lung vascular hyperpermeability in sepsis contributes to acute respiratory distress syndrome (ARDS), with no specific prevention or treatment strategies. Disruption of the microvascular endothelial cell (MVEC) barrier is a critical pathological feature of sepsis-induced lung injury driven by circulating inflammatory mediators, oxidants, and proteolytic enzymes. Our group has shown that plasma cell-free hemoglobin (CFH), released during sepsis due to red blood cell fragility, is a mechanistic driver of acute lung injury through induction of lung MVEC hyperpermeability. However, the cellular and molecular mechanisms are unknown. One potential mechanism by which CFH may disrupt the MVEC barrier is through its known ability to oxidize low-density lipoprotein (oxLDL). OxLDL binds and signals through its major endothelial receptor lectin-like oxidized LDL receptor 1 (LOX-1) to cause endothelial dysfunction. LOX-1 activation has been implicated in cardiovascular diseases such as atherosclerosis but its involvement in MVEC hyperpermeability during sepsis is unknown. Our preliminary data from patients with sepsis show that circulating CFH and oxLDL are tightly correlated with each other, MVEC injury markers, and mortality. However, little is known regarding LOX-1 receptor signaling leading to hyperpermeability, especially in the context of sepsis-induced injury to the lung microvasculature. This proposal aims to test the central hypothesis that MVEC hyperpermeability and lung injury during sepsis are mediated through oxidation of LDL by CFH to induce LOX-1 receptor signaling and ectodomain shedding.

NSF Awards · FY 2024 · 2024-08

Life as we know it hinges on the recapture of energy from the environment and its subsequent utilization in cellular processes. Consequently, the task of generating artificial “designer” mitochondria, the power plants in nearly all animal cells, is central to synthetic biology. This project will provide fundamental knowledge that is crucial for the ultimate goal of building artificial mitochondria, which can in turn be used in synthetic cells, and potentially in biotechnology applications and medical interventions. This project will also provide crucial training for the next generation of scientists and the STEM workforce of the future. Mitochondria stand out among animal organelles due to their possession of a cellular genome, similar to the nucleus, known as mitochondrial DNA. This DNA encodes several genes that are crucial for the most efficient cellular process of energy production. Therefore, the function of mitochondria is heavily reliant on their DNA, highlighting the pivotal role of our ability to maintain and manipulate DNA in this organelle for the goals of synthetic biology. Regrettably, our current understanding of mitochondrial DNA is so basic that reconstituting its replication in a test tube or even in a closely related organism (e.g., monkey mitochondrial DNA in human cells) presents insurmountable challenges. This represents a critical gap in our knowledge for creating artificial mitochondria. To address these issues, our preliminary studies introduced the GeneSwap approach, a genetic system enabling in situ reverse genetic analysis of proteins involved in mtDNA replication. Using this approach, the first protein controlling the species-specificity of mtDNA replication was identified. Additionally, for the first time, human/mouse somatic hybrid cells stably maintained human mtDNA. These new tools provide a unique resource to address critical questions in synthetic biology, focusing on the mechanisms of mtDNA replication and the species-specificity of this process. Therefore, in the proposed studies, we aim to build upon this initial success, identifying all factors required for human mtDNA replication and those contributing to its species-specificity as the first step toward generating artificial mitochondria. In the proposed studies, we will focus on three specific Aims: In Aim 1, a double-pronged approach will be used to identify new proteins contributing to the species-specificity of mtDNA replication. In Aim 2, replication of human mtDNA in mouse cells will be reconstituted by engineering them to express a defined set of human genes. In Aim 3, a structure-function analysis of the first identified component of IBMDR will be conducted to get mechanistic insights into IBMDR operation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-07

This project will contribute to development of a diverse, globally and locally (central area of the Gulf Coast) competitive semiconductor workforce, including women and other underrepresented minorities. In particular, the project will (1) increase strong partnerships and collaborations (both domestic and international) between academia, industry, and others; (2) improve and impact education and training of the advanced semiconductor workforce of the future; (3) align and incorporate industrial, professional, and technical standards in teaching and learning, thereby enabling participating students to have clear and smooth career pathways; (4) integrate systematic approaches to advance inclusive and equitable semiconductor education practices; (5) build capacity for the University to respond rapidly to changes in the workforce needed by the semiconductor industry; and (6) investigate student success in academia and in the semiconductor industry and associated fields. In addition, this project will practice and apply experiential learning pedagogy in emerging technology workforce exploration and demonstrate the effectiveness of the experiential learning theory in promoting and enhancing semiconductor workforce development. The Experiential-learning-based Undergraduate Semiconductor Workforce Exploration (E-USem) team will leverage strong industry-academic partnerships to advance and support the development of a skilled semiconductor workforce. Fundamental contributions and innovations to be developed by the team include: (1) Use of Kolb’s experiential learning theory to strengthen the workforce exploration and implement evidence-based instructional and inclusive practices. (2) Conducting seven unique experiential learning activities through collaboration between industry and academia. The E-USem team will first undertake core work such as development of the semiconductor course package; based on the course package, the team will create and develop a new Semiconductor Engineering concentration and Certificate program, a curriculum-sharing program, a summer program, and a Bridge Program. An Electronic Design Automation tool will be developed as well. (3) Systematically embedding diversity, equity, inclusion, and accessibility in the proposed E-USem activities from student recruitment, educational program design, to course design. This project aligns with the NSF ExLENT Program, as it seeks to support experiential learning opportunities for individuals from diverse professional and educational backgrounds to increase their interest in, and their access to, career pathways in emerging technology fields. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Highly recurrent and metastatic triple-negative breast cancer (TNBC) is predominant among young African American (AA) compared to European American (EA) women. Although mitochondrial DNA alterations play an essential role in tumor recurrence and metastasis, the mitochondrial genetic basis of TNBC racial disparity remains largely unknown. Mitochondria are unique organelles within the cells (having their own DNA) and are an integral part of the oxidative phosphorylation system (OXPHOS) for generating cellular ATP. They are composed of five complexes (I-V), which are assembled from multiple polypeptides- some encoded by mtDNA and others by nuclear DNA (nDNA). The human mtDNA is a 16.5-kb double-stranded closed circular molecule, which codes for the 12S and 16S rRNAs, 22 tRNAs, and 13 proteins essential for the mt respiratory complex (RC). Most human cells contain hundreds of copies of mtDNA and nearly all these mtDNA copies are identical, i.e., homoplasmic at birth as the mtDNA follows a strict maternal mode of inheritance. The mutation rate in mtDNA is approximately 10 times higher than that of the nuclear DNA and much easier to detect in cancer cells because of the high copy number. Studies from our lab and others have identified somatic mtDNA mutations in various cancers and demonstrated their role in cancer progression, implicating a role of mtDNA mutation in human tumorigenesis. Extracellular vesicles (EVs) harboring nucleic acids, proteins, and lipids are important determinants of tumorigenesis and promising for biomarker development. However, a comprehensive analysis of mtDNA alterations in triple-negative breast cancer racial disparity and their potential in noninvasive biomarker development remains largely unknown. In Aim 1, we will examine the pattern of the pre-optimized panel of 11- mtDNA mutations in EA and AA-TNBCs. This approach will enable us to validate the aggressive tumor- signature mtDNA mutational events and to better predict metastatic recurrence early and guide treatment planning. In Aim 2, we will measure the above panel of mtDNA mutations in the circulating EVs of all the EA and AA patients of Aim 1. Highly sensitive and accurate detection of the tumor-signature mtDNA mutations in the circulation as a function of progression will allow us to detect invasive lesions early and better predict metastatic recurrence and monitor therapeutic response. In the longer run, this research will lead to the development of routine clinical assays to detect disparate tumor-signature hotspot mtDNA mutations and measure mtDNA contents in extracellular vesicles for early diagnosis, recurrence prediction and treatment planning of the racially disparate population.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT The contribution of oxidative mitochondrial (mt) damage to inflammatory lung diseases is undisputed, but mechanisms underlying this association remain to be elucidated. Emerging evidence, however, points to the mitochondrial (mt) genome as a signaling hub integrating the initiation and resolution inflammatory responses. For example, one of the earliest events is reactive oxygen species (ROS) stress-induced oxidative mtDNA damage. Oxidized fragments of the mitochondrial genome (ox-mtDNA DAMPs) are then released into the cytosol where they stimulate the recognition receptor (PRR) NLRP3 to activate caspase-1 which triggers generation of proinflammatory cytokines. The mitochondrial base excision DNA repair (BER) pathway is important to this process since increasing or decreasing BER capacity suppressed or accentuated, respectively, mtDNA damage and ox-mDNA DAMP mobilization. BER also may be important in resolution of the inflammatory response. Full recovery requires restoration of mitochondrial function, accomplished in part by mitochondrial biogenesis. Mitochondrial DNA replication is a prerequisite for biogenesis, but replication must not take place until the mutagenic base damage initiating inflammation is repaired. Thus, BER and mtDNA replication must be faithfully coupled; if not, the resulting somatic mtDNA mutations would cripple recovery, potentially leading to acute and post-acute complications. Despite these advances, events underlying the transition between inflammation and its resolution are unclear. We now propose to test the hypothesis that a signaling axis involving mtDNA repair and caspase-1 integrates processes essential for initiation and resolution of NLRP3-dependent inflammation. Studies in cultured cells and intact rodents will: (1) Determine how BER coordinates initiation and resolution of ox-mtDNA DAMP formation with the intensity of inflammatory stress; and (2) Test the hypothesis that caspase-1 differentially regulates mtDNA repair and replication to ensure that NLRP3-induced inflammation is appropriate for the severity of the initiating stimulus while ensuring error-free, recovery-related mtDNA replication. This work is significant because it will test a new hypothesis to explain how the inflammatory response initiated by NLRP3 is titrated to the severity of the initiating stimulus and resolved without complications, including the potential introduction of somatic variants into mitochondrial genome during resolution of inflammation. These studies are also technically innovative; they will apply new mtDNA sequencing and isotopic labeling strategies to track the formation and fate of proinflammatory ox-mtDNA as it relates to lung cell inflammation and resolution.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY/ABSTRACT Our recent publications described pathogenic effects of the Pseudomonas aeruginosa type III secretion system effector ExoU on caspase-1 regulated inflammation. Our new preliminary data indicate that ExoU bypasses the inflammasome to induce a non-canonical form of caspase-1 activation in lung endothelial cells. However, the mechanisms underlying ExoU-directed caspase-1 activation are unknown. The consequences of ExoU-directed caspase-1 activation on a form of cell death known as pyroptosis are also undefined. ExoU is a phospholipase A2 (PLA2) that directly interacts with the host cell plasmalemmal membrane to induce lysis. We have discovered that ExoU also activates an indirect lysis pathway involving the gasdermin D (GSDMD) executioner of pyroptosis. Together, the data raise the intriguing prospect that ExoU-induced cell damage involves a combination of direct (ExoU PLA2 activity) and indirect (GSDMD-mediated) lysis pathways. The ExoU-induced indirect lysis pathway represents a novel virulence mechanism that contributes to P. aeruginosa pathogenesis. Based on our published and preliminary data, two complementary Specific Aims will test the Hypothesis that ExoU elicits non-canonical caspase-1 activation and processing of GSDMD to incite lung endothelial cell pyroptosis during P. aeruginosa infection. Aim 1 will elucidate mechanisms underlying ExoU-induced caspase-1 activation. Proposed experiments will determine whether: 1) ExoU PLA2 increases cytosolic Ca++ to stimulate calpain protease activation and 2) ExoU- mediated calpain activation liberates caspase-1 from the cytoskeleton to induce auto-activation. Aim 2 will examine the role of ExoU in GSDMD activation by: 1) rigorously validating the role of GSDMD in the ExoU indirect lysis pathway and 2) determining the role of ExoU PLA2 activity in GSDMD activation. The studies proposed herein are highly significant. P. aeruginosa is the most frequent Gram-negative, opportunistic pathogen causing pneumonia in patients with chronic lung disease (e.g., chronic obstructive pulmonary disease and cystic fibrosis), older age, and/or immunocompromised status. P. aeruginosa is also prevalent in critically ill patients with respiratory failure in the intensive care unit. Importantly, ExoU-expressing strains associate with the highest levels of patient morbidity and mortality. Thus, combined therapies targeting both ExoU and caspase-1 represent a pharmacological strategy to treat the most severe cases of P. aeruginosa induced pneumonia, acute lung injury, and sepsis. Moreover, the discovery that ExoU induces non-canonical caspase-1 and GSDMD activation, which drives lung cell death and dysfunction during P. aeruginosa infection is conceptually innovative. Our proposed studies will use technically innovative gene editing and inducible expression technologies to demonstrate cause-and-effect relationships between ExoU PLA2 activity, caspase- 1, and GSDMD towards testing our hypothesis.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Neutrophil activation in response to infection is a double-edged sword that can either kill invading pathogens and/or inflict tissue damage. Thus, neutrophils inextricably define whether the innate immune response to infection is beneficial or deleterious to the host. The interdependence of neutrophil degranulation and release of extracellular traps (NETs) has emerged as an important player in acute and chronic inflammation. This new R21 proposal is based on the unexpected discovery that the amyloid precursor protein (APP) regulates neutrophil degranulation and NETosis during Pseudomonas aeruginosa infection in the lung. While APP is known to drive the pathology of Alzheimer's disease via production of neurotoxic β-amyloid peptides, a growing body of evidence highlights an interplay between APP, β-amyloid, and innate immunity. Mice lacking APP are more susceptible to bacterial meningitis, and β-amyloid is an antimicrobial peptide. Preliminary data are presented to support the premise that App knockout mice show increased mortality and lung injury compared to wild type controls in response to P. aeruginosa infection. Surprisingly, P. aeruginosa-infected App knockout mice also exhibit increased neutrophil influx compared to wild type controls. In addition, in vitro studies demonstrate that isolated bone marrow-derived neutrophils from App knockout mice display increased degranulation and NETosis. Together, these published and preliminary data support a conceptually innovative and technically feasible approach for two Specific Aims that will test the HYPOTHESIS that APP modulates neutrophil degranulation and NETosis to limit lung injury during P. aeruginosa infection. Aim 1 will elucidate the protective role of APP during P. aeruginosa-induced lung injury. Aim 2 will test the utility of APP and β-amyloid peptides as predictors of outcome in critically ill patients. Our discovery that APP protects the host during P. aeruginosa lung infection is a highly significant conceptual advance with broad impact across the fields of lung biology and neurobiology. P. aeruginosa is the most frequent Gram-negative pathogen causing pneumonia in patients with chronic lung disease (e.g., chronic obstructive pulmonary disease and cystic fibrosis), and is prevalent in critically ill patients with respiratory failure in the intensive care unit. In the most severe cases, pneumonia progresses to acute lung injury, sepsis, and multi-organ failure. Importantly, survivors often suffer long-term sequelae such as post-intensive care syndrome (PICS) and neurocognitive dysfunction that reduce overall quality of life. Thus, our proposed studies may reveal potentially transformative links between a pathogen-mediated dysfunctional APP response in neutrophils and organ dysfunction and neurocognitive sequelae.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT This basic science grant seeks to determine fundamental mechanisms as to how a physiologically relevant cell controls the subcellular location of a signal to optimize the effect of that signal on cell function. We propose to build on recent findings by our diverse investigative team to delineate how 3 different Gs-coupled receptors in airway smooth muscle (ASM) cells - the 2AR, EP2, and EP4 receptors- have their signals compartmentalized within the cell to regulate important ASM cell functions. Aim 1 will employ novel imaging approaches to delineate spatiotemporal features of cAMP/PKA signaling by each receptor and demonstrate how this compartmentalized signaling is shaped by receptor-specific complements of AKAP and PDE isoforms, and by the competing co- activated Gq-coupled receptor. Aim 1 will also employ multiple subcellular-targeted biosensors to characterize the capacity of each receptor to signal from intracellular membrane compartments. Aim 2 will assess how these different receptors generate unique phosphoproteome signatures, and how manipulating the mechanisms shaping localized cAMP/PKA signaling regulates these signatures. Aim 3 will establish how the mechanisms dictating spatiotemporal features of ASM cAMP and the ASM proteome affect Gs-coupled GPCR regulation of ASM contraction, migration, and synthetic functions. The proposed studies will provide a foundation for understanding compartmentalized signaling in the form of both methodological advances and the knowledge gained in how Gs-coupled receptors employ distinct signaling mechanisms to render efficient and specific functional effects. From a translational perspective, our findings will constitute a critical basic science foundation for developing new drugs that target mechanisms of signaling compartments, most readily applied to better control asthma features such as airway hyperresponsiveness, airway remodeling, and possibly airway inflammation.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT The alveolar-capillary membrane facilitates efficient gas exchange while maintaining a restrictive permeability barrier. Pseudomonas aeruginosa infection disrupts the alveolar-capillary barrier leading to exudative edema and impaired oxygenation. P. aeruginosa utilizes a type III secretion system and its effectors to disrupt barrier integrity. In particular, the exoenzyme Y is introduced into lung endothelium, where it acquires nucleotidylyl cyclase activity and produces cGMP, cAMP, and cUMP. These cyclic nucleotide monophosphates activate protein kinase A resulting in endothelial tau phosphorylation, tau dissociation from microtubules, and microtubule breakdown, which collectively hinders repair following infection. Phosphorylated tau is released from endothelium as cytotoxic variants that contribute to lung injury. The signaling mechanisms used by exoenzyme Y to produce cytotoxic tau is incompletely understood, yet cUMP is produced at especially high concentrations and the cUMP signal parallels the generation of cytotoxic tau. Elevations in cUMP are sufficient to promote the production of cytotoxic tau variants. Our preliminary data demonstrate that the exoenzyme Y-induced cUMP signal also decreases endothelial nicotinamide adenine dinucleotide (NAD+) and increases nicotinamide, the product of NAD+ hydrolase activity, which may impair recovery following infection. Lung endothelium expresses sterile alpha and TIR motif containing 1 (SARM1), the only TIR (Toll/Interleukin-1 Receptor) domain protein in mammalian cells that possesses NAD+ hydrolase activity. Recent studies revealed a SARM1 bacterial homologue is directly activated by cUMP as an essential innate immune mechanism. While our studies illustrate an important role for cUMP in the endothelial cell response to infection, how exoenzyme Y generates the cUMP that leads to tau phosphorylation and production of cytotoxic tau variants, and how cUMP lowers NAD+ while hindering endothelial cell repair remains poorly understood. To address this knowledge gap in a rigorous way, this project tests the hypothesis that the P. aeruginosa exoenzyme Y generates cUMP, which contributes to the tau phosphorylation, microtubule breakdown, and SARM1-dependent NAD+ hydrolase activity that causes lung injury and hinders repair.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY & ABSTRACT Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the bunyavirus, SFTS virus (SFTSV). SFTSV is transmitted by the Haemaphysalis longicornis tick, which is native to East Asia but recently established invasive populations in the United States and continues to expand in geographic range. The rising incidence of SFTS cases in Asia, lack of specific treatment strategies, high case fatality rates, and global range expansion of the tick vector make SFTS a public health concern. As a tick-borne virus, SFTSV is unique from single-host viruses because it must replicate and survive in both vertebrate and invertebrate hosts. Currently, there is a critical need to elucidate the intra-tick and intra-host infection dynamics that enable bunyaviruses to infect, disseminate, and persist within the distinct environments of the tick and vertebrate host. Acquiring this fundamental knowledge is paramount to developing novel strategies that prevent SFTSV transmission. This research proposal is in direct response to NIH RFA-AI-21-046, “Promoting Bunyavirales Basic Science Research.” The overall objective is to define the dynamics of SFTSV infection, dissemination, and cell tropism within the tick vector, as well as the tick-to-host transmission timeline and initial SFTSV–host interactions in the skin. The central hypothesis is that biotic factors associated with H. longicornis’ life cycle facilitate intra-tick SFTSV dissemination to the salivary glands after molting, which in turn enables the tick to rapidly transmit SFTSV to the next vertebrate host on which it feeds while creating an immunologically privileged microenvironment at the skin site of tick feeding. The central hypothesis will be tested by pursuing two specific aims: 1) Characterize the dynamics of SFTSV infection, dissemination, and transstadial survival within H. longicornis ticks; and, 2) Define the minimum tick-to-host transmission time of SFTSV and the early host cutaneous immune response to SFTSV-infected tick feeding. Completion of these aims will define the infection kinetics and cell tropism of SFTSV within the tick vector across multiple life stages and within the skin of the vertebrate host. The proposed studies will be the first to collectively examine the intra-tick and intra-host infection dynamics of a tick-borne bunyavirus. Elucidating the fundamental H. longicornis–SFTSV–vertebrate host interactions will enable future work towards the development of rational interventions that disrupt virus survival within, and transmission between, the tick and vertebrate host.

NIH Research Projects · FY 2026 · 2022-03

Project Summary Neisseria gonorrhoeae, the causative agent for the sexually transmitted infection gonorrhea, is responsible for over 800,000 infections annually in the U.S. and 78 million cases worldwide. Untreated or untreatable infections can lead to infertility, pelvic inflammatory disease (PID) in females, gonococcal arthritis in both sexes, and an increased risk of both contracting and transmitting HIV. Over the past several decades, the inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonococcal infections. Most alarmingly, resistance against the extended-spectrum cephalosporin (ESC) ceftriaxone poses a serious threat to public health. This situation requires an understanding of antibiotic resistance at the molecular level in order to enable design of new antimicrobials. ESC resistance of N. gonorrhoeae is conferred by mutated forms of penicillin-binding protein 2 (PBP2). In this application, we propose to elucidate the molecular mechanism of resistance, with the overarching hypothesis that mutations in PBP2 restrict the molecular dynamics of the protein. It builds upon our recent understanding of the interactions made by wild-type PBP2 when bound by ESCs and how conformational changes associated with binding and acylation appear restricted in PBP2 derived from ESCR strains. The investigation comprises three aims: Specific Aim 1 is a structure-function analysis of wild-type PBP2 to investigate the importance of specific interactions formed when PBP2 is bound and acylated by cephalosporins. In Specific Aim 2, we will elucidate how key mutations present in PBP2 from ESCR strains of N. gonorrhoeae reduce inactivation by cephalosporins while retaining sufficient biological function to support growth of the organism. Finally, Specific Aim 3 will examine the behavior of PBP2 variants in solution to determine whether mutations hinder protein dynamics. By revealing the molecular mechanisms of how mutations in PBP2 overcome the lethal action of β-lactams, these investigations will enable new strategies for the development of replacement anti-gonococcal agents.

NIH Research Projects · FY 2023 · 2022-02

Nutrient accelerates cellular aging processes through metabolic stress. The detrimental effects of nutrient overload to health span are partially mediated by mTORC1 (mechanistic Target of Rapamycin Complex 1), an evolutionarily conserved nutrient-sensing kinase that signals for increase in anabolic processes. mTORC1 activity has been directly linked to aging and age-associated diseases in a diverse range of organisms including humans, mice, flies and worms. Remarkably, genetic or pharmacological inhibition of mTORC1 improved the health and increased the lifespan of several animal models of premature aging. Although the molecular mechanisms for mTORC1 activation by amino acids and growth factors are well established, recent findings indicate that excess glucose stimulates mTORC1 signaling through unconventional mechanisms that are not completely understood. Glucose metabolism directly and indirectly stimulates the production of the small metabolite inositol hexakisphosphate (IP6). Recent structural studies revealed that IP6 is tightly associated with mTOR, the catalytic subunit of mTORC1. Preliminary data suggest that IP6 binding to mTOR stabilizes the in vitro association between mTOR and RAPTOR, the regulatory subunit of mTORC1. The goal of this proposal is to establish a role for IP6 in the regulation of mTORC1 signaling in vivo and to assess whether targeting the metabolic pathways for IP6 synthesis will prevent cellular aging and promote longevity. In specific aim 1, the impact of IP6 metabolism on mTOR signaling and cellular ageing will be investigated. IP6 synthesis will be manipulated by suppression of the two critical kinases that catalyze the synthesis of IP6 – IPMK and IPK1. In addition we will suppress ISYNA1, the enzyme that catalyzes de novo synthesis of inositol from glucose. The direct effects of cellular IP6 on mTORC1/2 complex assembly and stability will be examined using recombinant mTOR mutants that are unable to bind to IP6. In specific aim 2, the crosstalk between IP6 metabolism and mTORC1 signaling will be genetically tested using C. elegans as a model for assessing longevity. Epistasis studies will be performed to determine how IPMK and IPK1 interact with of mTORC1. Suppression of enzymes involved in IP6 synthesis are predicted to protect worms from mTORC1- induced premature aging. Understanding the impact of IP6 on mTORC1 signaling and in aging will open up new opportunities for targeting these pathways to improve health.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY/ABSTRACT The proposed research plan focuses on improving our understanding of the effects of acidosis on pneumonia and establishing the conceptual basis for diagnostic and therapeutic translation. Acidosis is common in critically ill pneumonia patients, and is associated with high mortality. The pathophysiology of acidosis in pneumonia is poorly understood, and current therapies fail to improve major outcomes. Our studies have shown that pulmonary microvascular endothelial cells (PMVECs) utilize the carbonic anhydrase IX (CA IX) isoform to regulate pH, metabolism and migration. We also demonstrated that Pseudomonas aeruginosa infection of PMVECs induces release of cytotoxic amyloid proteins, which disrupts the alveolar capillary membrane. These cytotoxic amyloids induce soluble CA IX shedding from PMVECs which compromises their repair potential. Based on these preliminary studies, we test the hypothesis that P. aeruginosa infection induces cytotoxic amyloid production that leads to shedding of soluble CA IX in PMVECs, increasing lung injury. Specific aims test the hypotheses that: 1) CA IX is critical to the acid regulation, metabolism and migration of PMVECs and pulmonary endothelial barrier integrity; and, 2) P. aeruginosa infection elicits cytotoxic amyloid production, causing CA IX shedding in PMVECs, which increases lung injury. In vitro, we will use genetic approaches and endothelial cell functional assays to evaluate the effects of acidosis and the role of specific CA IX functional domains during physiologic and infectious conditions. In vivo and ex vivo, we will use acidosis, pneumonia and isolated lung perfusion mouse models to translate in vitro findings. Successful completion of this study will provide new insights into the mechanisms underlying acidosis in pneumonia and help identify CA IX and cytotoxic amyloids as biomarkers and therapeutic targets for pneumonia.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT The endothelium is a crucial regulator of vascular homeostasis and endothelial dysfunction is a hallmark of cardiovascular disease. The challenge in searching for new therapies is finding early control points that prevent the shift to broad pathologic signaling profiles and disrupt the endothelial network. Employing novel imaging and analysis approaches, we have identified discrete patterns of dynamic Ca2+ signalling along the vascular intima that underlie vascular function and direct the specificity, sensitivity and intensity of prevailing vascular responses. These patterns, defined by profiles of dynamic event parameters (frequency, amplitude, duration and spatial spread), form distinct signatures along the endothelial network. The complex spectrum of endothelial Ca2+ events (from isolated brief transients to broad multicellular waves) result from positive feedback interaction between plasma membrane TRP channels (Ca2+ entry) and endoplasmic reticulum IP3Rs (Ca2+ release). Small conductance Ca2+-activated K+ channels (KCa) play a key role in this signaling by exerting Ca2+-dependent hyperpolarization and amplifying Ca2+ influx through TRP channels (particularly fluid shear stress (FSS)- activated TRPV4 channels). In flow-deprived distal arteries from patients with peripheral artery disease, the endothelium exhibits a distinctive truncated Ca2+ signature characterized by spatially restricted small amplitude transients. This anomalous Ca2+ profile appears early in a low-flow carotid ligation mouse model, giving rise to endothelial dysfunction and vascular remodelling. These low-flow adaptations involve progressive loss of endothelial KCa2.3 channels and suggest an early loss of cooperative KCa/TRPV4 action. We hypothesize that disruption of TRPV4-KCa2.3 signaling under conditions of low FSS causes a progressive, highly restricted endothelial Ca2+ signature that promotes endothelial dysfunction and vascular remodeling. Aim 1 will characterize the role of TRPV4-KCa2.3 signaling in physiologic Ca2+ signatures along the arterial endothelium. We will conduct confocal imaging (with novel high-content analysis) and employ endothelium- specific knockout mice (ecKCa2.3-/- and ecTRPV4-/-) as well as human peripheral arteries to elucidate cooperative channel impacts under differential FSS. Aim 2 will determine whether low/oscillatory FSS causes truncation of the TRPV4-KCa2.3-dependent endothelial Ca2+ signature that leads to endothelial dysfunction and vascular remodeling. We will employ a partial ligation mouse model to assess the magnitude and time course of TRPV4- KCa2.3-specific impacts on Ca2+ signaling, vasoreactivity and vascular wall thickening. Aim 3 will determine whether preservation of endothelial TRPV4-KCa2.3 Ca2+ signaling ameliorates development of functional and structural vascular changes resulting from chronic low flow. We will also assess whether interventions to preserve the Ca2+ signature directly abate pathologic impacts of low flow.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY/ABSTRACT Patients in intensive care units are at high risk for long-term health threats including cognitive impairment. The correlation was only recently revealed after large-scale follow-up cognitive assessments on intensive patient survivors after their discharge from the hospital. There are testimonials, reviews and calls-to-action on many critical care websites and in journal issues over the last decade on this public health crisis. Studies have implicated delirium as a good predictor for long-term cognitive deficit; however, the causative and molecular mechanisms leading to abrupt cognitive impairment are unclear. In the past 4 years, our studies have discovered that patients in the intensive care unit who contracted bacterial pneumonia have elevated levels of cytotoxic amyloids in the bronchoalveolar lavage fluid, plasma, and the cerebrospinal fluid. Rodent brain slices incubated in the cerebrospinal fluids collected from bacterial pneumonia-positive patients show dampened hippocampal long-term potentiation. In comparison, synaptic strengthening is prominent in slices incubated in bacterial pneumonia-negative patients’ cerebrospinal fluid. Moreover, immunopurified from the cerebrospinal fluid or plasma using selective antibodies against Aβ and 𝜏 oligomers and injected into rodents, these cytotoxins induce neuronal dendritic spine retraction, reduce spine density, and impair animal learning. Our previous in vitro studies have implicated that in response to Pseudomonas aeruginosa infection, lung endothelium produces and releases cytotoxins including Aβ and 𝜏 species, and the cytotoxicity and bioactivity of these species are dependent upon the bacterial virulence. These endothelium-derived cytotoxins damage endothelial barrier integrity, hinder vascular repair following injury and, importantly, they are released into the systemic circulation in vivo. Thus, in this competitive renewal, the studies are designed to test the hypothesis that bacterial pneumonia-elicited lung endothelium-derived amyloids include pathological Aβ and 𝜏 species capable of dissemination and initiating aggregation. This work addresses a novel mechanism underlying the end organ dysfunction by systemically quantify cytotoxins released from endothelium in vitro, in rodents, and in patient specimens.

NIH Research Projects · FY 2025 · 2015-12

Rickettsia felis was originally identified in the United States as a human pathogen in 1991 and is now associated with human infection in North and South America, Europe, Africa, Asia, and Oceania. Our ultimate goal for this research is to elucidate the biological and molecular mechanisms that are critical to rickettsial transmission by fleas in order to better understand the epidemiology of flea-borne rickettsial diseases and identify novel points of intervention. Recent discoveries including transmission of R. felis in the absence of a rickettsemic host and the identification of multiple flea-borne rickettsial agents cocirculating in flea populations have guided the research to determine if sympatric rickettsial agents circulating in flea populations influence the transmission one another. Additionally, the assembly of the cat flea genome now allows for investigation of the flea-derived factors that facilitate or prevent Rickettsia transmission. The experimental focus of this proposal is to delineate horizontal transmission mechanisms through comparative analyses of coinfections using three distinct rickettsial strains in cat flea transmission bioassays. The flea-derived molecules associated with the transmission of Rickettsia by flea hosts are not known. Studies will also employ gene-editing in fleas to identify transmission determinants in a flea transmission system. Two limiting factors for vector/disease management and the barriers to advancing the field are the scant knowledge of 1) basic transmission biology of R. felis and other rickettsial pathogens in the context of coinfections and, 2) the flea-derived determinants of transmission. The need to overcome these barriers is evident as field collected fleas in areas of flea-borne rickettsioses outbreaks have multiple rickettsial agents circulating in the vector population and the fleas are known to be vectors of a number of pathogens, thus providing knowledge with a broad impact. Through completion of the specific aims outlined in this proposal, these studies will overcome the hurdles by delineating the role of coinfections in the transmission of R. felis (Specific Aim 1) and through identification of flea-derived molecules regulated in response to rickettsial infection relates to vector competence (Specific Aim 2). Thus, this is a multifaceted approach to decipher the vector and pathogen-associated factors essential to transmission and will provide a platform to examine other flea-borne bacterial pathogens.