VCOM Louisiana Research Day Program Book 2024

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ON THE COVER: (left to right) Randy Nguyen, OMS-II; Wasif Syed, OMS-II

Kareem Abdelhamid OMS-II

Welcome

Naved Salim OMS-III

Welcome to the VCOM Via Research Recognition Day on the VCOM Louisiana Campus. Each year, the Via Research Recognition Day is a significant event for VCOM that supports the mission of the College to provide medical education and research that prepares globally-minded, community-focused physicians and improves the health of those most in need. The Via Research Recognition Day offers a forum for health professionals and scientists in academic institutions, teaching hospitals and practice sites to present and benefit from new research innovations and programs intended to improve the health of all humans. By attending the sessions with guest speakers, participants have the opportunity to learn cutting edge information in the physiological bases of osteopathic manipulative therapy efficacy, new trends in physician-based research networks, and how to develop innovative research projects with high impact for human health. Poster sessions allow participants to learn about the biomedical, clinical and education-simulation research activities at VCOM-Louisiana and its partner institutions. This year, the Biomedical Research category at the VCOM-Louisiana Research Day event has been expanded to include two sections: Section 1 will feature presenters primarily from VCOM, while Section 2 will showcase contributions from our partner institutions. This change reflects rapidly growing interest and participation in this event.

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Agenda

8:00 a.m. – 9:00 a.m.

Registration, first floor Continental Breakfast, first floor, Room 107

9:00 a.m. – 9:15 a.m.

Opening Remarks Lecture Hall 2, second floor

9:15 a.m. – 10:15 a.m.

KEYNOTE LECTURE: “The Tale of an Insect-Specific Virus: from Ecology to Vaccine Development” Jonathan Auguste, PhD Virginia Tech Lecture Hall 2, second floor

10:15 a.m. – 10:30 a.m.

Coffee Break Second floor hallway

10:30 a.m. – 11:30 a.m.

KEYNOTE LECTURE: “Do Biomarkers Exist in the Thoracolumbar Fascia for LBP Pain?” Albert Kozar, DO, FAOASM, R-MSK VCOM-Virginia Lecture Hall 2, second floor

11:30 a.m. – 12:00 p.m.

PLENARY LECTURE: “The State of Research at VCOM” P. Gunnar Brolinson, DO, FAOASM, FAAFP, FACOFP Lecture Hall 2, second floor

12:00 p.m. – 1:00 p.m.

Lunch First floor, Room 107

12:30 p.m. – 2:30 p.m.

Poster Competition Rooms-130-132, first floor

2:30 p.m. – 3:30 p.m.

Award Ceremony and Final Remarks Lecture Hall 2, second floor

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RESEARCH

changes the world.

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Contents

Speakers

Program Moderators.................................................................................................................................................................6

Plenary Speaker.........................................................................................................................................................................9

Keynote Speakers....................................................................................................................................................................10

Abstracts

Biomedical Research: Section 1.............................................................................................................................................12

Biomedical Research: Section 2.............................................................................................................................................27

Clinical Research.....................................................................................................................................................................41

Case Studies............................................................................................................................................................................59

Education, Simulation and Best Practices.............................................................................................................................83

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Program Moderator

Dixie Tooke-Rawlins, DO, FACOFP President VCOM

Dr. Dixie Tooke-Rawlins has served as the administrative officer principal to the founding of the Edward Via Virginia College of Osteopathic Medicine in 2001. As the founding dean for VCOM, she led the College in the development of the curriculum, clinical site affiliations, budget and long range plan, and assisted in the design of the first educational and research facilities. She currently holds the position of President, as well as the academic title of professor in the Department of Family Medicine at VCOM. In 2011, Dr. Tooke-Rawlins again served as a founding Dean in the opening of the Carolina campus of VCOM in Spartanburg, South Carolina, and again in 2015 in the opening of the Auburn Campus of VCOM in Auburn, Alabama. Prior to this appointment, Dr. Tooke-Rawlins held the position of interim dean at Kirksville College of Osteopathic Medicine (KCOM) in Kirksville, Missouri. Past appointments include associate dean for academic and clinical affairs at KCOM, director of osteopathic medical education and program director of the Osteopathic Family Practice Residency at St. Luke’s Hospital in Allentown, Pennsylvania,

and family practice residency director at Metropolitan Hospital in Grand Rapids, Michigan. She currently holds several appointments nationally and the state level including president of the Virginia College of Osteopathic Family Physicians, Governor appointee to the Virginia Council on Healthcare Reform and the Virginia Healthcare Workforce Development Authority. Dr. Tooke-Rawlins is a member of the Board of Governors of the American Association of Colleges of Osteopathic Medicine, and has served on the inspection teams for both osteopathic post-graduate training institutes and osteopathic family medical residencies. She is a board member of the American Osteopathic Foundation, and she has been a member of the American Osteopathic Association and the American College of Osteopathic Family Physicians for more than 20 years. Dr. Rawlins has focused her career on the interests of osteopathic medicine, osteopathic medical education, rural health/rural medicine, health care disparities, and global health. She

was instrumental in the establishment of the VCOM international medical missions program that provides year round primary care clinics in Honduras, El Salvador and the Dominican Republic. She participates in annual outreach programs in Appalachia including remote community outreach programs by the college and has for many years served the uninsured and medically underserved with time committed to community service. Dr. Tooke-Rawlins is a graduate of Northeast Missouri State (now Truman State University) and the Kirksville College of Osteopathic Medicine with a Doctorate of Osteopathic Medicine degree. She completed her post graduate work at Grandview Hospital in Dayton, Ohio and entered the field of emergency medicine for the first seven years of her early career. She returned to Kirksville where she entered the field of family medicine and became board certified in family medicine by the American Board of Osteopathic Family Practice.

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Program Moderator

Mark A. Sanders, DO, JD, MPH, LLM, MS, FACOFP, FACLM Dean, Louisiana Campus VCOM

Mark A. Sanders, DO, JD, MPH, LLM, MS, FACOFP, FACLM, is the dean for The VCOM (VCOM)'s Louisiana campus. Before becoming Dean, Sanders held the position of associate dean for curriculum, assessment and medical education for VCOM-Louisiana, a role he had previously held at VCOM's Auburn campus. He continues to teach genetics, geriatrics and ethics at VCOM. His research interests include topics in geriatrics, elder law, transitional care, chronic disease and molecular genetics. He specializes in family medicine, geriatric medicine, legal medicine and public health. He specifically caters to homebound elderly individuals and other adults with disabilities. Sanders graduated from Texas Christian University with a degree in Biology/Chemistry. He then obtained his medical degree from UNTHSC-TCOM and his law degree from Texas A&M College of Law (formerly Texas Wesleyan University). He then received a Master's in Public Health degree from UNTHSC-SPH and a Master's in Elder Law from Stetson University College of Law. Most recently, he obtained a graduate degree in clinical molecular genetics

from Northern Michigan University. Sanders is a fellow in the American College of Osteopathic Family Practice and in the American College of Legal Medicine. In addition, he serves on the Textbook Editorial Committee for LEGAL MEDICINE 2023, written by the American College of Legal Medicine. He authored a chapter in the same textbook on the use of artificial intelligence in genetics. Sanders is married to Anada Gunn-Sanders, JD, MPH, and they have three children: Sofia, Liam and Emma Grace.

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Program Moderator

Pawel Michalak, PhD Associate Dean for Biomedical Affairs & Research, Louisiana Campus VCOM

Pawel Michalak, PhD; serves as as the Associate Dean for Biomedical Affairs & Research for VCOM (VCOM)'s Louisiana campus. He is also an adjunct faculty member at the Virginia-Maryland College of Veterinary Medicine and the University of Haifa in Israel. Michalak previously served as a professor at the VCOM-Virginia campus since 2017, with a focus on comparative genomics, bioinformatics, and One Health, operating as head of the Integrative Genome Analysis Lab (IGAL) at the Center for One Health Research. His team has employed an integrative approach with a variety of molecular, computational and modeling techniques, including NextGen sequencing of entire genomes, epigenomes, and transcriptomes to establish rigorous understanding of how complexity elaborates from genomes through gene regulatory networks, and how it evolves over generations in response to environmental stress and intragenomic challenges.

Michalak has worked at nine universities in four countries across three continents. He has published his research in one book, with a second currently in preparation. Additionally, Michalak has been credited as a contributor in multiple books, as well as approximately 70 peer-reviewed papers in scientific journals, including “Cell,” “Science,” “Nature,” “Proceedings of the National Academy of Sciences USA” and “Proceedings of the Royal Society.” His research has been cited nearly 2000 times and popularized by various public media outlets.

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Plenary Speaker

P. Gunnar Brolinson, DO, FAOASM, FAAFP, FACOFP Vice Provost for Research VCOM

Dr. Brolinson is vice provost for research, professor of family and sports medicine at the VCOM and team physician for Virginia Polytechnic Institute and State University. He is an adjunct professor in the Department of Mechanical Engineering at Virginia Tech. He is the fellowship director emeritus of the Primary Care Sports Medicine Fellowship at VCOM and Virginia Tech. He is also a volunteer physician for the United States Olympic Committee and a team physician for the United States ski team and was head team physician for the freestyle ski team at the 2006 Winter Olympic Games in Torino, Italy. He was also named to the medical staff for the 2010 Winter Olympic Games in Vancouver, BC, and was medical director of the USOC performance services center. He obtained his undergraduate training from the University of Missouri at Columbia earning a degree in biology. A 1983 graduate of the Kirksville College of Osteopathic Medicine, Dr. Brolinson is board certified in family practice and holds a subspecialty certification in sports medicine.

Prior to coming to Virginia, he was the co director of the Primary Care Sports Medicine Fellowship training program at The Toledo Hospital in Toledo, Ohio and team physician for University of Toledo. He has extensive experience in undergraduate and post-graduate medical education. Dr. Brolinson has served on the boards of the American Osteopathic Academy of Sports Medicine, the American Medical Society for Sports Medicine and the Midwest Chapter of the American College of Sports Medicine. He is a past president of the American Osteopathic Academy of Sports Medicine. Dr. Brolinson is a fellow of the American Osteopathic Academy of Sports Medicine, the American Academy of Family Practice and the American College of Osteopathic Family Practice. In 1997 he was named outstanding young physician in Ohio by the Ohio State Medical Association. He is a former associate editor for the Clinical Journal of Sports Medicine and a former member of the editorial board of the Physician and Sports Medicine. He is former section editor

for Competitive Sports and Pain Management in the journal Current Sports Medicine Reports. Dr. Brolinson is a frequent speaker at national sports medicine meetings and often teaches didactic laboratory sessions on the use of osteopathic manipulative therapy for athletic injuries. Dr. Brolinson is a contributing author in the latest edition of Foundations for Osteopathic Medicine and he has published several scholarly articles and book chapters in the area of sport and exercise medicine. His research interests have included exercise and immune function, exercise and bone mineral density, mild traumatic brain injury in sports, impact biomechanics, human factors in auto safety, sports performance and manipulation and other health and disease prevention related topics.

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Keynote Speaker

Jonathan Auguste, PhD Associate Professor of Arbovirology Virginia Tech

A. Jonathan Auguste, PhD, is an associate professor in the field of Arbovirology at Virginia Polytechnic Institute and State University. Dr. Auguste currently serves as the director for the Fralin, BSL3/ACL3 labs, and he is the co-director of the Infectious Diseases Interdisciplinary Graduate Education Program. Auguste’s research focuses on arbovirus surveillance, understanding the mechanisms by which insect-specific viruses are unable replicate in vertebrate cells, and elucidating the mechanisms by which these viruses preclude super-infection of pathogenic flaviviruses in vivo. He is also developing novel viral-vectored vaccine platforms against important arboviruses within the Flavivirus and Alphavirus genera. More recently, he has focused his efforts developing new animal models and creating mRNA vaccines for endemic Orthobunyaviruses that are important to public and veterinary health. He has also contributed to the development of several SARS-CoV-2 vaccines and therapeutics, including one candidate that is in use in Argentina. Auguste has a very active research program and is funded by an NIH R01

and R21 and also by the USDA and HHMI. Auguste has authored or co-authored more than 50 peer-reviewed articles on arbovirus research. He also runs a relatively large program and his lab contains several graduate students, postdocs and technicians. He is very committed to graduate education and is currently serving on advisory committees for 13 graduate students at Virginia Tech only.

Auguste is also an expert salsa dancer.

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Keynote Speaker

Albert J. Kozar, DO, FAOASM, R-MSK Professor for Osteopathic Manipulative Medicine and Sport Medicine; Director of the ONMM Residency VCOM

Albert J. Kozar, DO, FAOASM, R-MSK, is the HCA/VCOM ONMM3 residency director and professor for OMM and Sports Medicine at VCOM-Virginia. He is a fellow of American Osteopathic Academy of Sports Medicine and is board certified in ONMM, Sports Medicine and Family Medicine. Dr. Kozar is a registered musculoskeletal sonographer, and since coming to VCOM-Virginia, he has developed multiple research lines looking at tendonopathy, TLF in LBP and visceral motion with OMT. Dr. Kozar was honored as the 2022 VCOM Researcher of the Year for VCOM-Virginia. Dr. Kozar’s interests lie in neuromuscular control of functional rehabilitative approaches, diagnostic and therapeutic uses of musculoskeletal ultrasound, OMT and regenerative medicine therapies. He is a regular national lecturer for multiple academies on these topics.

Prior to joining VCOM-Virginia in the fall of 2015, he was in private practice in Avon, Connecticut, was a team physician for The University of Hartford, Bloomfield High School, and covered multiple professional sporting events nationally.

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Biomedical Research: Section 1

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Biomedical Research: Section 1

Dan Luu, OMS-II; Keith Jackson, PhD; Dinesh Aryal, PhD VCOM-Louisiana; ULM College of Pharmacy 1 ASSESSMENT OF RENAL CHANGES DURING METABOLIC ACIDOSIS PROMOTED HYPERTENSION

Background: Chronic metabolic acidosis (CMA) evoked hypertension has been recently delineated in experimental rats. The synergistic effect of intrarenal angiotensin II (Ang II) and superoxide (a free radical component) has been depicted as a cause of the observed hypertension. Therefore, treating acidotic rats with captopril (ACE inhibitor) and tempol (superoxide scavenger) in combination has shown to be effective in controlling high blood pressure. In the present study we investigate the magnitude of renal changes that occurred during acidotic, acidotic hypertensive and acidotic normotensive states in rats. Objective: To evaluate the magnitude of renal changes occurred during chronic acidosis elicited hypertension compared to acidotic normotensive conditions. Methods: Male Sprague Dawley rats (100-150 g) were divided into three groups: (i) Control (ii) CMA (acidotic), (iii) CMA+captopril+tempol. CMA was induced by providing a weak acid solution of 0.28 M ammonium chloride in drinking water for 8 weeks. Blood pH was measured to confirm rats were acidotic, while blood pressure was monitored weekly using tail-cuff. Urine samples were collected to measure creatinine, albumin-urea, sodium and potassium levels.

Glomerular filtration rate (GFR) was analyzed among all three groups. Further, urine NGAL level was analyzed to assess the kidney injury. Hematoxylin and eosin (H&E) staining were used to verify histological details in the cortical kidney sections. Results: Results demonstrated a significant decline in GFR in the CMA rats vs control group. However, GFR in CMA+captopril+tempol treated acidotic rats is significantly increased vs CMA rats, which suggested that the reduced GFR in CMA group is because the rats are hypertensive. Conversely, the urinary albumin excretion was significantly elevated in acidotic rats but the CMA+captopril+tempol treated group showed a significant reduction in albuminuria, which revealed the acidosis-induced hypertension is contributing for impaired renal infiltration and augmenting the albuminuria. The NGAL levels were significantly increased in CMA rats but declined in CMA+captopril+tempol treated group. The H&E staining revealed significant structural changes with signs of dilated tubular lumina, cellular detachment, and an absence of nuclei indicating tubular necrosis in CMA rats. However, when these rats were treated with captopril and tempol combined, the glomeruli were intact, and nuclei were seen evenly distributed.

Conclusions: Overall, our results suggested the renal consequences of acidosis induced hypertension can lead to eventual kidney injury which is independent of the function of acidosis. Moreover, intrarenal Ang II and superoxide release in the kidney could be a potential target to prevent or treat acute kidney injury (AKI) and its further progression. Further studies are needed to examine if Ang II and superoxide directly affect the renal blood flow during the pathological acute renal injury.

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Biomedical Research: Section 1

Malvina Kartamyshev, OMS-II; Hassan Khanani, OMS-II; Roopin Singh, OMS-II; Abir Islam, OMS-II; Yao Liang, OMS-II; Kareem Abdelhamid, OMS-II; Wasifuddin Syed, OMS-II; Murtaza Khambhati, OMS-II; Tanya Kumar, OMS-III; Dalal Dawud, BPharm; Zakaria Abd Elmageed, PhD VCOM-Louisiana 2 SYNERGISTIC EFFECT OF CAMBINOL AND APALUTAMIDE ON METASTATIC CASTRATION-RESISTANT PROSTATE CANCER CELLS

Background: Metastatic castration-resistant prostate cancer (mCRPC) poses a significant clinical challenge due to its resistance to conventional therapies. Targeting multiple pathways is one of the effective options to manage patients with mCRPC. Objective: In this study, we explored the potential synergistic effects of Cambinol, a specific inhibitor of neutral sphingomyelinase 2 (n-SMase 2), and Apalutamide, a potent antagonist of phosphorylated AKT (phospho AKT), on mCRPC cell lines; CWR-R1ca and PC-3M. Methods: Our investigation employed a multidimensional approach, combining MTT assays, colony formation assays, scratch assays, and Western blot analysis to comprehensively evaluate the therapeutic effect of the drug combination. Results: The MTT assay revealed a notable reduction in cell viability upon treatment with the combined Cambinol and Apalutamide, indicating a synergistic anti-proliferative effect. Colony formation assays further demonstrated a significant suppression of clonogenic potential, suggesting a potential impairment of the cells’ ability to form colonies. Scratch

assays elucidated a pronounced inhibition of cell migration, emphasizing the potential role of the combined drugs to impede metastatic processes in mCRPC cells. To elucidate the molecular mechanisms underlying these observed effects, Western blot analysis shows that Cambinol demonstrated an inhibition of sphingomyelinase activity, while Apalutamide effectively suppressed phospho-AKT activity. Conclusions: Our findings provide compelling evidence for the synergistic anti-cancer effects of Cambinol and Apalutamide on mCRPC cells, highlighting the potential of this combination as a promising therapeutic strategy. This study suggests a dual-targeted approach to enhance therapeutic efficacy for treatment of patients with mCRPC.

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Biomedical Research: Section 1

3 THE ROLE OF SER-214 PHOSPHORYLATED TAU IN LUNG ENDOTHELIAL HOST DEFENSE

Grace Corrier, OMS-II 1 *; Kassady Perkinson, OMS-II 1 *; Elizabeth Turnage, OMS-II 1 *; Ron Balczon, PhD 2,3 ; Chung-Sik Choi 3,4 , PhD; Mike T. Lin, PhD 3,4 ; Troy Stevens, PhD 3,4,5 ; Rebekah Morrow, PhD 6 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 * *Indicates equal contributions 1 Department of Cell Biology & Physiology, VCOM-Louisiana 2 Department of Biochemistry, University of South Alabama College of Medicine; 3 Center for Lung Biology, University of South Alabama College of Medicine; 4 Department of Physiology & Cell Biology, University of South Alabama College of Medicine; 5 Department of Internal Medicine, University of South Alabama College of Medicine; 6 Department of Microbiology & Immunology, VCOM-Louisiana

Background: Nosocomial pneumonia is a significant concern in the critical care setting. Lung infection promotes the release of pro inflammatory mediators and cytotoxins into both the distal airways and pulmonary circulation. Patients that recover from nosocomial pneumonia often suffer lasting physical and cognitive impairments post-discharge. Less than one third of those discharged will survive the first year following release. Endothelial derived hyperphosphorylated tau has recently been identified as one of the cytotoxins that comprise the inflammatory milieu generated by lung infection. Infection elicited tau is highly cytotoxic, self-propagating, and transmissible, constituting a tauopathy. Pseudomonas aeruginosa expressing a type III secretion system (T3SS) mediated toxin, exoenzyme Y (ExoY), is one of the most common agents of nosocomial pneumonia. ExoY intoxication of PMVECs induces hyperphosphorylation of lung endothelial tau at the Ser-214 residue which promotes increased permeability and a secondary proteinopathy. ExoY has also been reported to suppress the antimicrobial function of cytoprotective lung endothelial amyloid species. However, whether the contribution of endothelial tau and its phosphorylation status

contributes to this phenomenon remains poorly understood. Objective: ExoY generated endothelial tau is necessary to suppress innate amyloid function whereas Ser-214 phosphorylation significantly contributes to the ExoY mediated impairment of the host defense amylome. Methods: Wildtype and various genetically manipulated PMVECs were utilized for our studies including stable tau KO (global tau KO), control (tau KO carrying an empty plasmid), 1N4R (tau KO with plasmid encoding endothelial tau isoform 1N4R only), and S214A (tau KO harboring a plasmid encoding tau 1N4R with a serine-to-alanine point mutation at residue 214; S214A). PMVECs with plasmids were cultured in full media containing G418 for selection. Confluent monolayers of PMVECs were treated with either vehicle, a non-virulent strain of P. aeruginosa unable to inject ExoY into cells (∆PcrV), and an isogenic strain capable of injecting ExoY (ExoY+) alone into cells. PMVECs were infected with a multiplicity of infection of 20:1 in HBSS and incubated for 6 h. Supernatant fractions were collected at 6 h post-infection, filter-sterilized, and centrifuged in concentrators

with a 3 kDa MW cutoff. Concentrated supernatants were standardized to 0.5 mg/ml of protein in HBSS. Samples were then boiled and cold shocked to denature non-amyloid proteins prior to use in assays. Antimicrobicity was measured through Kirby-Bauer disk diffusion, kinetic bactericidal assays, aggregation assays, and Syto9/propidium iodide staining. Tau species were assessed via immunoblotting and amyloid burden was assessed with Thioflavin T, Congo Red birefringence, and immunoblotting. Results: TBD Conclusions: TBD

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Biomedical Research: Section 1

Zachary Dickey, OMS-III 1 , Kiara Pankratz, OMS-II 1 *, Annie Pham, OMS-II 1 *, Elizabeth Kibodeaux, OMS-II 1 *, Dara W. Frank, PhD 2 , Troy Stevens, PhD 3 , Rebekah Morrow, PhD 1 , K. Adam Morrow, PhD 1 , Sarah Voth, PhD 1 *Indicates equal contributions 1 VCOM-Louisiana; 2 Medical College of Wisconsin; 3 University of South Alabama Whiddon College of Medicine 4 SIRT1 ACTIVATION ABLATES TAU RELEASE DURING EXOY-COMPETENT PSEUDOMONAS AERUGINOSA INFECTION

Background: Pseudomonas aeruginosa is one of the most common agents of nosocomial pneumonia and acute respiratory distress syndrome (ARDS). While mortality rates in ARDS have improved, survivors often face significant chronic physical and cognitive impairment. P. aeruginosa strains responsible for acute infection of the distal airways usually have a syringe-and-needle type III secretion system (T3SS) that delivers a combination of up to 3 of a potential 4 toxins directly into the cytosol of host cells. The T3SS-mediated toxin most frequently utilized in acute lung infection is the nucleotidyl cyclase, exoenzyme Y (ExoY). ExoY produces marked disruption of interendothelial junctions, edema, and suppression of the innate immune response. Moreover, ExoY intoxicated endothelium generates the release of cytotoxic endothelial-derived amyloids including hyperphosphorylated tau. ExoY elicited amyloids propagate damage independently of the primary infection and transmit injury from cell-to-cell and host-to-host thereby constituting a proteinopathy. However, the mechanisms germane to ExoY induced cytotoxic amyloid release remains poorly resolved. The sirtuin 1 (SIRT1) NAD+ dependent deacetylase has been implicated in the regulation of the autophagic degradation of dysregulated amyloidogenic proteins. Thus, activating SIRT1 may present a viable option for alleviating the cytotoxic amyloid burden arising from lung infection. Objective: In this study, we tested whether pharmacologic activation of SIRT1 would reduce the

pathogenic tau burden elicited secondary to ExoY competent infection. Methods: Our studies utilized rat pulmonary microvascular endothelial cells (PMVECs). Bacteria included strains expressing either ExoY (ExoY+) alone or an isogenic activity-null mutant (ExoYK81M). PMVECs were infected at a multiplicity of infection (MOI) of 20:1 in HBSS. To ascertain the potential role of SIRT1, we identified a particularly selective SIRT1 agonist (SRT2104) with negligible affinity for other sirtuin proteins as well as a highly selective SIRT1 inhibitor, EX-527. Infection experiments were conducted with and without SRT2104 and EX-527. Initial experiments were conducted over a range of concentrations for SRT2104 (1, 3, and 6 µM) and EX 527 (5, 10, and 20 µM). Infections were imaged over time to assess signs of toxicity and interendothelial disruption. To determine whether the drug killed the bacteria directly or conferred protection to the cells despite bacterial doubling, aliquots from ExoY+ infections were serially diluted and plated over time. Lysates and supernatants were collected at 0 and 6 h post-infection and probed for pathogenic tau harboring a phosphatase-activating domain (PAD) with the TNT1 antibody, amyloid oligomers (A11 antibody), and SIRT1. Supernatants were also filter-sterilized, boiled, ice shocked, and applied to naïve cells to determine the potential for transmissible cytotoxicity. Results: ExoY intoxication alone was sufficient to significantly impair levels of SIRT1 in whole cell

lysates while amplifying pathogenic tau export into the supernatant fraction. Activation of SIRT1 via treatment with SRT2104 during ExoY infection notably reduced intracellular tau and ablated ExoY generated PAD-bearing tau in the extracellular space. SIRT1 inhibition (EX-527 treatment) during infection with the activity null mutant ExoYK81M was sufficient to increase intra- and extracellular PAD tau as compared to the untreated control. Tau was not observed in ‘cell free’ infection experiments. ExoY generated oligomeric amyloid constituents in the supernatant fraction were likewise abrogated with SIRT1 agonist SRT2104. In transmissibility experiments, neither the vehicle- nor ‘cell free’ derived supernatants induced injury. However, heat-stable transmissible cytotoxins generated from ExoY competent induced significant injury, toxicity, and cell death. Damage from boiled/ shocked supernatants from SRT2104 treated ExoY+ infections of PMVECs were commensurate with those from cyclase-null control strain ExoYK81M infection. Conclusions: ExoY intoxication of PMVECs suppresses endothelial SIRT1 and disrupts the turnover of pathogenic amyloid species to promote PAD-harboring tau production and release. Moreover, SIRT1 activation during ExoY-competent infection mitigates the burden of PAD in PMVECs and protects downstream endothelium from secondary amyloid/ tau mediated injury. Thus, the agonist SRT2104 may provide a clinically relevant means of preventing the acute secondary injury and long-term sequelae common to survivors of nosocomial pneumonia/ARDS.

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Biomedical Research: Section 1

Lucia Conlon, BS, OMS-II; Janika Ernst, BS, OMS-III; Claire Travis, MS, OMS-II; Lin Kang, PhD; and Melissa Lipsmeyer, MS, PhD VCOM-Louisiana 5 EXPLORING THE POTENTIAL PROTECTIVE ROLE OF THE BACTERIAL-DERIVED METABOLITE INDOLE-3-PROPIONIC ACID AGAINST OBESOGENIC INSULTS IN PLACENTA CELL LINES

Background: Obesity during pregnancy is associated with detrimental changes in placental function that lead to the development of complications such as gestational diabetes and preeclampsia as well as altered metabolism in the offspring. With the incidence of obesity reaching epidemic levels world-wide, it is imperative to find mechanisms to mitigate these risks during pregnancy. The gut-microbiome is a diverse system that regulates numerous body functions through the secretion of specific metabolites. Gut dysbiosis has recently emerged as one mechanism that contributes to placental dysfunction and clear associations between obesity and gut dysbiosis are evident. Indole 3-propionic acid (IPA) is a gut microbiome derived metabolite that has anti-inflammatory, anti-oxidative stress and glucose regulatory functions, all of which are dysregulated in obesity. Previous studies have found altered levels of IPA in both serum and placentas of women with overweight/obesity and that serum IPA levels are correlated with lower levels of adiposity, circulating insulin and insulin resistance. However, it remains unknown what the physiological functions of IPA are in the placenta and if it could have potential protective effects against perturbations caused by obesity. Objective: The objective of this study is to

determine what physiological role IPA plays in the placenta and if IPA can protect against obesogenic insults such as hyperinsulinemia, oxidative stress, lipid peroxidation and inflammation which contribute to placental pathology. We hypothesize that physiological levels of IPA will reduce inflammatory markers, alleviate oxidative stress and modulate glucose uptake through altered insulin signaling in placental cells. Methods: For this study, we developed an in vitro model of obesity where two human placental cell lines, BeWo (choriocarcinoma) and HTR8 (normal immortalized trophoblasts) were cultured in media conditioned by mature human adipocytes (ACM). RNA sequencing (RNAseq) analysis was used to determine acute effects of IPA treatment after 24 hours in the presence and absence of ACM. We further examined the influence of IPA on insulin signaling with physiological and hyperinsulinemia levels of insulin at 24 and 72 hours utilizing QPCR and western blot analyses. Results: RNAseq analysis revealed treatment with physiological levels of IPA induced changes in genes associated with normal placental function, autophagy and inflammatory mediated processes and other pathways. Subsequent

studies on the impact of IPA on insulin signaling, glucose uptake and oxidative stress are ongoing. Conclusions: Our RNAseq analysis offers initial insight into the molecular mechanisms of IPA in placental cells. Future analyses will investigate the potential role of insulin signaling, glucose homeostasis and oxidative stress. Collectively, this study highlights the importance of a healthy gut microbiome and potentially serves as a window of opportunity to mitigate the onset or severity of pregnancy-related disorders such as gestational diabetes by enhancing the gut microbiome prior to pregnancy.

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Biomedical Research: Section 1

Annie Pham, OMS-II; Kiara Pankratz, OMS-II; Zachary Dickey, OMS-III; Elizabeth Kibodeaux, OMS-II; Erika Lytle, OMS-III; Dara W. Frank, PhD; Troy Stevens, PhD; Rebekah Morrow, PhD; K. Adam Morrow, PhD; Sarah Voth, PhD VCOM-Louisiana; Medical College of Wisconsin; University of South Alabama Whiddon College of Medicine 6 EXOY INTOXICATION SUPPRESSES LUNG ENDOTHELIAL TAK1 TO PROMOTE TAUOPATHY

Background: Nosocomial pneumonia is of particular concern in the critical care setting. Acute opportunistic infection of the distal lung promotes edema, hypoxia, and acute respiratory distress syndrome (ARDS). Both nosocomial pneumonia and ARDS are associated with poor outcomes and reduced longevity among survivors post-discharge. After release, many suffer an acute-to-chronic disease transition with marked functional impairments both physically and neurologically. The Gram negative nosocomial pathogen Pseudomonas aeruginosa is the predominant agent responsible for nosocomial pneumonia and ARDS. Over 90% of virulent clinical isolates of P. aeruginosa utilize a syringe-and-needle like type III secretion system (T3SS) to inject the nucleotidyl cyclase toxin exoenzyme Y (ExoY) into the cytosol of target cells. ExoY generates unconstrained cyclic nucleotides with subsequent breach of the lung air-blood barrier and subversion of the host’s innate immune defense. ExoY has been reported to suppress the innate immune response via inhibition of transforming growth factor- β -activating kinase 1 (TAK1) in some cell types. Pulmonary microvascular endothelial cells (PMVECs) intoxicated with ExoY release virulent tau cytotoxins that propagate injury and are transmissible among species indicative of a prion

strain. In our studies, we have sought to clarify the role of TAK1 in the production of ExoY-generated cytotoxic tau. Objective: We hypothesize that ExoY intoxication of PMVECs inhibits TAK1 to promote production of pathogenic tau. Methods: For infection studies we used isogenic strains of P. aeruginosa expressing 1) an incompetent T3SS needle (∆PcrV; cannot form the translocation pore required to facilitate toxin injection), 2) an activity-null mutant unable to generate cyclic nucleotides in the host cell (ExoYK81M), and a mutant that injects only virulent ExoY (ExoY+). Secondary rat pulmonary microvascular endothelial cells (PMVECs) were infected at a multiplicity of infection (MOI) of 20:1 in HBSS and incubated for 5 hours. Vehicle and ‘no cell’ infection wells were used as controls. Infected cells were imaged over time to assess signs of toxicity and endothelial barrier disruption. Whole cell lysates and cell supernatants were collected at 0 and 5 h post infection. Subsequently, lysates and supernatants were probed for TAK1, endothelial tau harboring a phosphorylation-activating domain (PAD) using the TNT1 antibody, and p65 NF-kB. For lysates, protein concentration was determined using a standard BCA assay and 30 µg of protein

was loaded for each sample. Blots of lysates were normalized to GAPDH. Supernatants were precipitated using TCA-DOC, loaded by volume, and normalized to total protein. Cadaveric Alzheimer’s hippocampal lysate and recombinant proteins were used as controls. Results: In lysates, TAK1 and p65 NFkB were elevated above vehicle for control strains ∆PcrV and ExoYK81M infected cells. However, TAK1 levels were abrogated in ExoY intoxicated cells. ExoY+ infection depressed p65 NFkB in PMVECs. TNT1-reactive tau was elevated above vehicle in ExoY intoxicated cell lysates and increased nearly three-fold in the supernatant fraction collected from ExoY+ infected PMVECs. No TNT1 signal was detected in ‘no cell’ infection controls. Activation of upstream sirtuin 1, a known regulator of TAK1 mediated signaling, rescued TAK1 activity in ExoY infected cells and ablated endothelial derived PAD-harboring tau release. Conclusions: These data implicate ExoY in the suppression of the TAK1 immune regulatory protein in lung endothelium. Moreover, our results shed light on the signaling pathway(s) germane to ExoY-induced pathogenesis. Taken together, our findings suggest that ExoY likely inhibits sirtuin 1 to constrain TAK1 modulated innate immunity and generate endothelial derived tau cytotoxins.

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Biomedical Research: Section 1

Rachita Gupta, BS, OMS-II; J. Annelies Hayward-Smithey, BS, OMS-II; Riva Kelly, BS, OMS-II; Melissa Lipsmeyer, MS, PhD VCOM-Louisiana 7 INVESTIGATING THE FUNCTION OF THE BACTERIAL-DERIVED METABOLITE INDOLE-3-PROPIONIC ACID IN ENDOMETRIAL CANCER

Background: Endometrial cancer is the most common malignancy of the female reproductive tract and its incidence and mortality are rising. Obesity is the most common risk factor for the development of endometrial cancer and is thought to drive pathological transformation and proliferation of the endometrium by increasing circulating estrogen levels, hyperinsulinemia and inflammation. Recent evidence has suggested that obesity-driven dysregulation of the gut microbiome contributes to the metabolic perturbations observed in endometrial cancer. The gut bacteria-derived metabolite indole-3 propionic acid (IPA) has anti-inflammatory and glucose regulatory properties in various normal tissues and some cancer types. However, no studies have evaluated the effects of IPA on endometrial cancer. Objective: The goal of this study is to determine the potential protective effects of IPA on endometrial cancer cell proliferation and viability as well as the impact of IPA on insulin signaling and glucose uptake in endometrial cancer cells. Given that endometrial cancer is largely driven by excess estrogen and impeded by progestins, we will evaluate the effects of IPA on estrogen and progesterone signaling to determine if IPA can prevent estrogen-driven proliferation or enhance the inhibitory actions of progesterone.

Methods: For this study we are utilizing the well-differentiated human endometrial adenocarcinoma cell line, Ishikawa. This cell line retains expression of both estrogen and progesterone receptors, making it ideal to discover novel interactions between the gut microbiome and sex steroid receptor signaling. Because endometrial cancer pathogenesis is largely driven by obesity, we will be utilizing our in vitro model of obesity where cells are cultured in media conditioned by human adipocytes in order to mimic an obesogenic environment. The effects of IPA on cell viability will be assessed by MTT assay. QPCR and western blot analysis will be utilized to investigate the impact of IPA treatment on steroid hormone receptor and insulin signaling pathways. Mechanistic assays evaluating glucose uptake will also be performed to complement studies on insulin signaling. Results: Analyses from this study are currently underway and are to be determined. Conclusions: This study will offer novel insights between the intersection of female reproductive health and the gut microbiome. Importantly, these investigations will reveal possible mechanism by which a disturbed gut microbiome can contribute to pathogenic changes of not only tissues of the female

reproductive tract, but of other tissues. Future studies will evaluate the effects of IPA on normal human endometrial stromal cells for a comparative analysis. Collectively, these studies will highlight the importance of a healthy gut microbiome which can likely be achieved through diet and healthy lifestyle modifications and serve as a window of opportunity to mitigate the onset of endometrial carcinogenesis in high risk women with overweight/obesity.

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Biomedical Research: Section 1

Melissa Ruiz, MS, OMS-III 1 *; Chung-Sik Choi, PhD 2 ; Mike Lin, PhD 3,4 ; Troy Stevens, PhD 3,4 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 1 Department of Cell Biology and Physiology, VCOM-Louisiana; 2 Department of Physiology and Cell Biology, University of South Alabama College of Medicine; 3 Center for Lung Biology, University of South Alabama College of Medicine; 4 Department of Internal Medicine, University of South Alabama College of Medicine 8 AMYLOID PRECURSOR PROTEIN CONTRIBUTES TO LUNG ENDOTHELIAL IMMUNE RESILIENCE

Background: Innate immune resilience refers to a tissue’s ability to maintain functional integrity during a microbial insult and to repair itself following resolution of the active infection. The innate resilience of the air-blood barrier is fundamental to preventing alveolar flooding and hypoxemia during nosocomial pneumonia. Bacterial breach of the endothelial barrier promotes permeability, bacteremia, and acute respiratory distress syndrome (ARDS). The inflammatory milieu characteristic of ARDS is partially comprised of endothelial-derived cytotoxic amyloids. Pathogenic amyloids elicited from infected lung endothelium contribute to the long-term cognitive and physical impairments that describe post-intensive care syndrome. Cytotoxic amyloids arise from the corruption of their innate physiologic function by specific bacterial toxins. Physiologically functional amyloids – including amyloid-beta (A β ) – are innately bacteriostatic and bactericidal. Non-pathogenic A β is generated from the amyloidogenic processing of amyloid precursor protein (APP), which has been reported to contribute to vascular resilience in the cerebrovascular compartment. However, there has been little elucidation on the role of APPs in the resilience aspect of lung endothelial host defense. Objective: We hypothesize that APP promotes lung endothelial barrier resilience by regulating the endothelial immune response.

Methods: Rat PMVECs utilized in these studies included a wildtype (WT), CRISPR/Cas9 control (APP-expressing subclone from CRISPR/Cas9), and two subclones in which APP was deleted (APP-/-). Subconfluent PMVECs were lifted, and 100,000 cells were seeded into 6-well plates for growth curves to assess whether APP contributes to PMVEC proliferation. Three wells from each clone were lifted, counted, and counts averaged at the same time each day. Averages were plotted over time and the doubling time calculated. Wound closure assays were used to determine APP’s contribution to cell migration and endothelial barrier repair. For wound closure assays, PMVECs were grown to confluence in 6-well plates and a 100 µl pipette tip was used to scratch the monolayer to induce injury. Images were taken over time and analyzed with ImageJ to quantify wound area. RT-qPCR measured the basal level of pro-inflammatory mediators IL-6, MCP-1, TNF α , and CCL5. Angiogenesis assays were utilized to assess the potential role of APP in vascular network formation. Subconfluent PMVECs were identically seeded in Matrigel-containing 48-well plates in full media and imaged over time. ImageJ was used to quantify the number of closed loops, intersections, and distance between intersections of vascular networks. Results: Although there was no difference between the doubling time of WT and control PMVECs (ns;

p < 0.3500), the rate of APP-/- cell doubling was significantly impaired as compared to the control (p < 0.0027) and WT cells (n ≥ 3; 3 replicates, p < 0.0001; one-way ANOVA with Tukey’s post hoc). WT PMVECs demonstrated a decreased ability to repair a disrupted endothelial barrier as compared to APP-/- cells. No difference in wound area between WT, control or APP-/- PMVECs immediately following injury (ns; p < 0.8743, 0.5007, and 0.6677) was noted. At 18 h post-injury, however, WT PMVECs exhibited a decreased ability to repair the injury (p < 0.0166) as compared to both control PMVECs (p < 0.0106) and APP-/- cells (p < 0.8117). Moreover, no notable differences in angiogenic capacity (ns) or wound closure was noted between control and APP-/- PMVECs (p < 0.8117; n ≥ 3; 3 replicates, two-way ANOVA with Tukey’s post-hoc). Interestingly, APP null cells significantly overexpress cytokine CCL5 as compared to control PMVECs (p < 0.001) or WT (n ≥ 2, two-way ANOVA with Tukey’s post-hoc analysis) PMVECs. The dysregulation of CCL5 has been heavily implicated as integral to the development of neurodegenerative disease. Conclusions: APP is fundamental to lung endothelial barrier resilience by promoting cell proliferation, stability, and regulating the production of CCL5.

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Biomedical Research: Section 1

Sabeen Wazir, OMS-I; Brina Snyder, PhD; Thomas Floyd, MD VCOM-Louisiana; UTSW Medical Center 9 OPTIMIZATION OF PARADIGMS FOR ASSESSING COGNITIVE PERFORMANCE WITHIN A HYPOXIC ENVIRONMENT

Background: Chronic Obstructive Pulmonary Disease, anemia, and obstructive sleep apnea are examples of chronic hypoxic conditions that often adversely affect aging populations and are associated with cognitive impairment. The gold-standard for assessing cognitive outcomes has been behavioral testing paradigms in rodent models. However, their usage in understanding chronic hypoxic conditions has been hindered by the inability to perform these tests under stable hypoxic conditions. Changes to behavior tests impact the validity of the paradigm. Objective: We present validated testing paradigms that can be performed in hypoxic chambers to model hypoxia-inducing diseases and allow for better understanding of chronic conditions. Methods: 3 month old (3 mo) wild-type male and female C57Bl6/J mice (n=6 each sex) were housed, trained and tested within enclosed hypoxia chambers from Biospherix, Ltd. Spontaneous Alternation (SA) and Spatial Object Recognition (SOR) paradigms were selected as tests of working memory. Mice were housed and acclimated to specific oxygen (O2) conditions in one chamber and transferred to a separate chamber for testing under the same O2 level. Following one day of acclimation to either 21%

or 15% oxygen, protocols were conducted as outlined in D’Isa, et al., 2021 (SA) and Leger, et at., 2013 (SOR). Animal movements and interactions were tracked using an overhead camera and EthoVision tracking software. To determine validity, thresholds for chance were set at greater than 50% alternation over 7 trials for SA, and greater than 60% novel location and 0.25 D.I. for SOR. The total number of object contacts in a familiar and a novel location were quantified and used to calculate the percentage of contacts in the novel location and a discrimination index (D.I.) using the following formulas: % novel = (# novel contacts / total number of contacts) * 100 and D.I. = (# novel contacts - # familiar contacts) / total # contacts. Results: In SA, 3 mo mice exhibited a percentage of alternation 61.11%, which is above the expected threshold for chance. As expected, testing under 15% O2 reduced alternation to 33.33%. In the SOR, both the % novel (66.57%) and D.I. (0.33) were also above chance for 3 mo normoxic control mice. The results indicate both the SA and SOR paradigms can be adapted to assess behavior under mild moderate hypoxic conditions. Conclusions: In conclusion, these paradigms more accurately mimic cognitive performance

and functional conditions for people who suffer from chronic hypoxic conditions.

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Biomedical Research: Section 1

Kiara Pankratz, OMS-II; Annie Pham, OMS-II; Zachary Dickey, OMS-III; Elizabeth Kibodeaux, OMS-II; Erika Lytle, OMS-III; Dara W. Frank, PhD; Troy Stevens, PhD; Rebekah Morrow, PhD; K. Adam Morrow, PhD; Sarah Voth, PhD VCOM-Louisiana; Medical College of Wisconsin; University of South Alabama Whiddon College of Medicine 10 PSEUDOMONAS AERUGINOSA TOXIN EXOY CONSTRAINS LUNG ENDOTHELIAL AUTOPHAGIC FLUX TO PROMOTE TAU RELEASE

Background: The Gram-negative nosocomial pathogen Pseudomonas aeruginosa is the predominant agent responsible for nosocomial pneumonia and acute respiratory distress syndrome (ARDS). Patients frequently suffer poor physical and cognitive outcomes post discharge with less than 50% surviving the first year with that number following to one third in the following year. Over 90% of P. aeruginosa strain isolated from critically ill patients with acute nosocomial lung infection express a type III secretion system (T3SS) that works like a syringe-and-needle to inject a nucleotidyl cyclase toxin (ExoY) into the cell’s cytosol. ExoY generates unconstrained cyclic nucleotides within the host cell causing aberrant signaling and phosphorylation events. Intoxication of endothelium with ExoY at the air-blood barrier of host alveoli induces disruption of the endothelial barrier, alveolar flooding, and the production and release of hyperphosphorylated oligomeric tau into the extracellular milieu. ExoY generated endothelial derived tau is a heat-stable cytotoxin that can propagate from one cell to another and from one host to another with little loss of virulence. However, the mechanisms responsible for the generation of ExoY instigated pathogenic tau have not been resolved. Objective: We hypothesize that ExoY

constrains autophagic flux to promote the production and release of cytotoxic tau. Methods: Isogenic strains of P. aeruginosa expressing 1) an incompetent T3SS needle (∆PcrV; cannot form the translocation pore required to facilitate toxin injection), 2) an activity-null mutant unable to generate cyclic nucleotides in the host cell (ExoYK81M), and a mutant that injects only virulent ExoY (ExoY+) were used to infect rat pulmonary microvascular endothelial cells (PMVECs). Cells were treated with either vehicle or infected at a multiplicity of infection (MOI) of 20:1 in HBSS and incubated for 5 hours. ‘No cell’ wells of infection suspension only were incubated in parallel with infected cells as a control. Infected cells were imaged over time to assess signs of toxicity and endothelial barrier disruption. Supernatants and whole cell lysates were collected at 0 and 5 h post-infection and probed for LC3-I and LC3-II and endothelial derived tau harboring a phosphorylation-associated domain (PAD) using the TNT1 antibody. Lysate protein concentration was measured using a standard BCA assay. The antibody signal of lysates was normalized to GAPDH. TCA-DOC was used to precipitate supernatant samples. Samples of supernatant were loaded by volume and normalized to total protein. Cadaveric Alzheimer’s hippocampal

lysate and recombinant proteins were used as controls. Results: LC3-I identifies the autophagosome (cargo proteins ATG3/7). Lipidation of LC3-I produces LC3-II and signifies the fusion of the lysosome with the autophagosome (autolysosome). Vehicle treated cells exhibited the most robust LC3-I to LC3-II conversion/ turnover with greater LC3-II density. PMVECs infected with nonvirulent control strains ∆PcrV and ExoYK81M displayed equivalent levels of LC3-I to LC3-II conversion. ExoY intoxicated PMVECs displayed significantly reduced autophagic flux with a marked reduction in both LC3-I and LC3-II. The TNT1-reactive tau burden was largely ablated in supernatant fractions collected from ExoY+ infected cells. Moreover, pharmacologic activation of sirtuin 1 rescued autophagic flux while concurrently ablating PAD tau in the extracellular milieu. Conclusions: Our findings strongly implicate ExoY-mediated inhibition of endothelial autophagic flux as a mechanism of pathogenic tau production and release in nosocomial P. aeruginosa pneumonia.

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