VCOM Louisiana Research Day Program

1 2023 Via Research Recognition Day 2023 Via Research Recognition Day

Welcome

Welcome to the inaugural Edward Via College of Osteopathic Medicine 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.

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Agenda

FRIDAY, APRIL 14, 2023

8:00 a.m. – 9:00 a.m.

Registration, first floor Continental Breakfast, first floor, main hall

9:00 a.m. – 9:15 a.m.

Opening Remarks Lecture Hall 2, second floor

9:15 a.m. – 9:45 am.

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

9:45 a.m. – 10:30 a.m.

KEYNOTE LECTURE: “Variants, Vaccines and Viral Genomic Surveillance. What Have We Learned from COVID-19?” Jeremy Kamil, PhD LSU Health Shreveport Lecture Hall 2, second floor

10:30 a.m. – 12:00 p.m.

Poster Session Room 107, first floor

12:00 p.m. – 1:00 p.m.

Lunch First floor

1:00 p.m. – 1:45p.m.

KEYNOTE LECTURE: “Neurosurgical – Anatomical Research: A 20-Year Journey” Jorge E. Alvernia-Silva, MD Brain and Spine Associates, LLC Lecture Hall 2, second floor

1:45 p.m. – 2:30 p.m.

Poster Competition Awards, Closing Remarks and Adjournment Lecture Hall 2, second floor

2:30 p.m. – 5:30 p.m.

Fish Fry Picnic – Live Music (“T Broussard & the Zydeco Steppers”) Bayou Patio

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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...............................................................................................................................................................12

Clinical and Case Studies........................................................................................................................................................25

COVID-19 and Epidemiology...................................................................................................................................................51

Education Research.................................................................................................................................................................59

Pharmacology..........................................................................................................................................................................64

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Program Moderator

Dixie Tooke-Rawlins, DO, FACOFP President and Provost Edward Via College of Osteopathic Medicine

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 and Provost 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.

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.

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

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Program Moderator

Mark A. Sanders, DO, JD, MPH, LLM, MS, FACOFP, FACLM Dean, Louisiana Campus Edward Via College of Osteopathic Medicine

Mark A. Sanders, DO, JD, MPH, LLM, MS, FACOFP, FACLM, is the Dean for The Edward Via College of Osteopathic Medicine (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 Edward Via College of Osteopathic Medicine

Pawel Michalak, PhD, serves as as the Associate Dean for Biomedical Affairs & Research for Edward Via College of Osteopathic Medicine (VCOM)'s Louisiana campus. 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 Edward Via College of Osteopathic Medicine

Dr. Brolinson is Vice Provost for Research, Professor of Family and Sports Medicine at the Edward Via College of Osteopathic Medicine 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

Jeremy P. Kamil, PhD Associate Professor of Microbiology and Immunology LSU Health Sciences Center Shreveport

Jeremy P. Kamil, PhD, is an Associate Professor of Microbiology and Immunology at LSU Health Sciences Center Shreveport (LSUHS). Dr. Kamil’s research has focused on human cytomegalovirus (CMV), a common virus that silently infects most people but is a leading cause of birth defects and of devastating opportunistic infections in immunosuppressed people. In March 2020, at the outset of the U.S. COVID-19 outbreak, Dr. Kamil spearheaded the establishment of a local viral genome sequencing program, which would grow to become the #1 source of variant surveillance data from the state of Louisiana. In fact, as of Jan. 30, 2023, the LSUHS Emerging Viral Threats Laboratory has shared 17,875 coronavirus genome sequences via GISAID, the major global sharing mechanism for COVID-19 variant data. Dr. Kamil has advocated for decentralized approaches to viral genomic surveillance, including rapid sharing of the resulting data. Recently, he led a Rockefeller Foundation grant to establish viral genome sequencing capacity at smaller, rural academic institutions, including historically Black colleges and universities.

Dr. Kamil’s work identifying coronavirus variants has been covered in national media outlets, such as the New York Times and CBS Evening News. He has also been interviewed on NPR about coronavirus variants, including a major segment on WNYC’s ‘On the Media,’ about the importance of properly crediting S. African, Botswana and Hong Kong who discovered the Omicron variant. Dr. Kamil’s major ongoing research focuses on how CMV manipulates glycobiology for immune evasion.

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Keynote Speaker

Jorge E. Alvernia-Silva, MD, FAANS American Board Certified Neurosurgeon Brain and Spine Associates, LLC

Dr. Alvernia is an American Board Certified Neurosurgeon who is internationally educated and trained. After graduating with honors from medical school, Dr. Alvernia completed his second neurosurgical residency training at Tulane Medical School in New Orleans, Louisiana, in 2010. Additionally, Dr. Alvernia completed his first neurosurgical residency training (Valedictorian) in Bogota, Colombia, in 2000. From 2001-2003 he completed a Skull base fellowship in Lyon, France, under the world-renowned Pr. Marc Sindou. Additionally, he completed a microsurgical anatomy research fellowship at the University of Illinois in Peoria, where he also served as the teaching faculty. He specializes in spine conditions, brain tumors, and nerve disorders. Dr. Alvernia has worked as a neurosurgeon in Fort Smith, Arkansas, and holds a medical license to operate in Arkansas and Louisiana. He has worked for the past eleven years in Northeast Louisiana and was honored with 2015, 2017, and 2018 Health Care Heroes/ Neurology-Neurosurgeon Awards for Northeast Louisiana. After four years as the sole Neurosurgeon for the regional medical

Glenwood Regional Medical Center, he moved into private practice (Brain and Spine Associates) in 2016. Additionally, he holds an appointment as a Neurosurgery Affiliate Faculty member in the Neurosurgery Department at the University of Mississippi. In that capacity, he works at the Research Microsurgical anatomy lab with the Neurosurgery residents. He has written over 50 publications in international peer-reviewed journals and several neurosurgery textbook chapters. His surgical anatomy drawings have been featured in renowned international journals’ cover pages, such as the Journal of Neurosurgery and Acta Neurochirurgica. Additionally, he serves as a section editor (Spine and Skull base) for WORLD NEUROSURGERY, WORLD NEUROSURGERY:X, and Ad Hoc reviewer for national and international neurosurgery journals and books. Dr. Alvernia is fluent in English, Spanish, and French. In addition, Dr. Alvernia serves as the president of the US chapter of the Colombian Neurosurgical society.

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Biomedical Research

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Biomedical Research

Olivia B. Bee, OMS-II; Rebekah L. Morrow, PhD; Stephen DiGuiseppe, PhD; Sarah B. Voth, PhD; K. Adam Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana 01 ALTERED NUCLEAR LOCALIZATION OF PULMONARY ENDOTHELIAL EXPRESSED TAR DNA-BINDING PROTEIN 43 IN RESPONSE TO PSEUDOMONAS AERUGINOSA INFECTION

Context: TAR DNA Binding Protein 43 (TDP 43) is a major component of pathologic inclusions in Amyotrophic lateral sclerosis (ALS), Frontotemporal Dementia (FTD) and is implicated in Alzheimer’s Disease (AD). TDP-43 proteinopathies in neurodegenerative diseases are characterized by loss of nuclear TDP-43 and accumulation of the ubiquitinated protein aggregates in the cytosol, with proteolytic cleavage of TDP-43 into abnormal C-terminal fragments. Recent evidence suggests roles for TDP-43 in immunity and inflammation, specifically related to the pathogenesis of such proteinopathies. While studied extensively in neuronal diseases, there is limited data on the role of TDP-43 in response to pulmonary endothelial injury. Previous work has established that Pseudomonas aeruginosa infection causes damage to pulmonary endothelial cell barriers, and that the strain of P. aeruginosa PA103 utilizes the T3SS effectors ExoU and ExoT, which has an established role in cellular injury and gap formation, in a manner characteristic of an endothelial proteinopathy. Objective: Here, we aim to establish cellular localization of TDP-43, and to determine the effect of P. aeruginosa , as a model of promoting cellular stress, on quantifiable levels, as well as location, of host expressed TDP-43.

Methods: Rat pulmonary microvascular endothelial cells were grown to confluence and infected with P. aeruginosa . Images of the cell monolayer were captured, and lysates were collected hourly during infection. TDP 43 levels were assessed via western blot and densitometry conducted using ImageJ. Separate infections were conducted across a range of multiplicities of infection (1:1 to 20:1), and immunofluorescent staining for TDP-43 was subsequently performed. Results: P. aeruginosa induces visible and time-dependent gap formation in endothelial monolayers, confirming previously published work establishing cellular injury in response to such infections. Western blot analysis shows that TDP-43 is decreased intracellularly in response to infection. Immunofluorescence of uninfected cells indicates nuclear localization of TDP-43. While still partially nuclear, there appears to be greater cytoplasmic signal, and less nuclear signal in response to infection, suggesting importance of further exploration of underlying pathophysiological cellular redistribution of TDP-43 in response to cellular stress. Conclusions: We conclude that Pseudomonas aeruginosa infection of pulmonary endothelial

cells results in diminished levels of TDP 43, and loss of nuclear TDP-43, consistent with pathologic alterations of the protein in neurodegenerative diseases.

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Biomedical Research

Zachary Dickey, OMS-II 1 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Chung-Sik Choi, PhD 2 ; Mike Lin, PhD 2 ; Troy Stevens, PhD 2 ; and Sarah Voth, PhD 1 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 University of South Alabama – Whiddon College of Medicine, Mobile, Alabama 02 THE ROLE OF SIRT1 IN THE PATHOGENESIS OF PSEUDOMONAS AERUGINOSA TOXIN EXOENZYME Y

Background: Nosocomial pneumonia describes the onset of pneumonia ≥ 48 hours post-admission and commonly arises in the critically ill. Pseudomonas aeruginosa is the most common cause of nosocomial pneumonia, which frequently gives rise to acute respiratory distress syndrome, sepsis, and consequent multiorgan damage. Sequelae often include long-term physical and cognitive impairment as well as increased rates of mortality with less than 50% of patients surviving the first year post discharge. Within the parenchyma of the lung, the alveolar epithelium and capillary endothelium are closely appositioned and joined by a shared basement membrane forming a thin respiratory barrier to facilitate efficient gas exchange. Virulent strains of P. aeruginosa possess a type III secretion system (T3SS) that functions like a needle and syringe to inject toxins directly into the host cytosol. Roughly 90% of P. aeruginosa clinical isolates utilize a T3SS-mediated nucleotidyl cyclase effector, exoenzyme Y (ExoY). ExoY intoxication generates unconstrained increases in cytosolic cyclic nucleotides (cNMP). In the endothelium of the pulmonary capillary (pulmonary microvascular endothelial cells, or PMVECs), ExoY primarily

microtubule collapse, and the production and release of oligomeric tau. However, the other mechanism(s) of ExoY pathogenesis are not well-understood. Normally, autophagy promotes the breakdown and clearance of dysregulated tau. In neurodegenerative disease, autophagic flux is inhibited and induces the accumulation and oligomerization of dysregulated tau in the endocytic pathway. Pathogenic tau oligomers are then exocytosed into the extracellular milieu where they propagate injury in a prion-like manner. The NAD+-dependent deacetylase sirtuin 1 (SIRT1) modulates mitochondrial function, autophagy, and innate immunity making it a likely target for ExoY-mediated inhibitory phosphorylation. Hypothesis: We hypothesize that ExoY intoxication inhibits lung endothelial SIRT1 to constrain autophagy and innate defense while promoting the production and release of pathogenic tau. Methods: To test our hypothesis, PMVECs will be infected with isogenic mutants of P. aeruginosa at an MOI of 20:1 both with and without pharmacologic inhibition/activation of SIRT1 over a range of concentrations. Exogenous caged cyclic nucleotide analogs (cGMP-AM and cUMP-AM) will be utilized in

the absence of infection to ascertain whether cytosolic increases in cNMPs alone are sufficient to inhibit SIRT1. Isogenic P. aeruginosa mutants include ∆PcrV (incompetent T3SS needle), ExoY K81M (ExoY incapable of cyclase activity), and ExoY + (injects ExoY only). Lysates will be collected over time and examined for 1) SIRT1 levels (immunoblot), 2) SIRT1 activation (immunoblot and ELISA), and 3) SIRT1 transcript (RT-qPCR). Cytokine release will be monitored via RT-qPCR and ELISA, autophagic flux will be assessed through probing lysates for LC3-I to LC3-II conversion, and tau release will be monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red birefringence. Cyclic nucleotides will be measured with ELISA. Results: TBD Conclusions: TBD

generates cGMP and cUMP, suppresses the innate immune response, induces

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Biomedical Research

Ariana E. Faraji, OMS-III; Kasia Michalak, MS; Lin Kang, PhD; Stephen DiGiuseppe, PhD; Pawel Michalak, PhD Edward Via College of Osteopathic Medicine, Monroe, Louisiana 03 INVESTIGATING THE ROLE OF INTERNALLY TRANSCRIBED SPACERS OF RIBOSOMAL RNA IN VIRAL INFECTIONS

Context: Ribosome biogenesis is the process of making ribosomes, which involves generating and processing of ribosomal RNAs (rRNAs). The 18S, 5.8S, and 28S rRNAs are transcribed into a single precursor, where each rRNA is separated by non-coding spacer regions. Between 18S and the 5.8S rRNA is Internal Transcribed Spacer (ITS) 1 and between 5.8S and 28S rRNA is ITS2. The spacers are released by endonuclease activity with average fragments sizes ranging from 150 – 250 for ITS2 detected in eukaryotic cells. Despite this, investigating biological roles of ITS fragments have been mostly ignored because they are presumed to be degraded. Interestingly, we recently discovered that eukaryotic 45S rRNA gene clusters have several striking Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-like features, such as palindromic organization of highly conserved sequences across species (18S, 5.8S, and 28S rDNA). In addition, we uncovered significant sequence homology within ITSs to a variety of viral genomes including the Alphaherpesvirus, herpes simplex virus 1 (HSV-1). It was recently reported that ribosome biogenesis restricts innate immune response to infection of a Betaherpesvirus, human cytomegalovirus (HCMV). Based on these recent findings, we hypothesize that ITS fragments

serve a hitherto unappreciated role in regulation of the innate immune response in the context of viral infections. Objective: To evaluate biological functions of ITSs in eukaryotic cells and their immune responses to viral infections. Methods: The ITS1 and ITS2 regions were sequenced using Sanger and Nanopore sequencing. We generated siRNA sequences targeting regions found across ITS1 and ITS2 sequences and a scramble control. We transfected siRNAs into normal dermal human fibroblasts (NHDFs). Next, NHDFs were infected at a low multiplicity of infection (MOI) with viruses from different viral families including KOS strain HSV-1, WR strain of vaccinia virus (VacV), and Indiana strain of vesicular stomatitis virus (VSV). Viruses were titered on BSC40 cells and viral proteins were detected via Western blot analysis from total protein lysates post infection. Results: Knockdown of ITS2 exhibited differential increases of titers for each virus tested when compared to scramble control siRNA (n = 3). We also detected differential increases in viral protein levels that agree with our viral titers.

Conclusion: Our preliminary data suggests that ITS2 fragments may serve a previously unappreciated role in restricting viral replication. Interestingly, not all ITS2 fragments demonstrated the same levels of restriction implying that there may be viral specificity. Whether that restriction is due to innate immune impairment is still unknown, but we plan to investigate the role of innate immune pathways after depleting ITS fragments in our future studies. Eukaryotic ITSs contain fragments of virus-like sequences that may play a role in restricting viral replication, which may provide insight into a new paradigm for antiviral innate immunity.

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Biomedical Research

Sabra Hanna, MLS(ASCP), OMS-II 1 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Brant Wagener, MD, PhD 2 ; Jean-Francois Pittet, MD 2 ; Chung-Sik Choi, PhD 3 ; Mike Lin, PhD 3 ; Troy Stevens, PhD 3 ; and Sarah Voth, PhD 1 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 University of Alabama – Birmingham, Heersink School of Medicine, Birmingham, Alabama; 3 University of South Alabama – Whiddon College of Medicine, Mobile, Alabama 04 PATHOGENESIS OF KLEBSIELLA PNEUMONIAE INDUCED PROTEINOPATHY IS AMYLOID STRAIN DEPENDENT

Background: Gram-negative opportunist Klebsiella pneumoniae has been designated a ‘Critical Priority’ pathogen by the World Health Organization due to its increasing prevalence in pneumonia and sepsis, as well as its evolving multidrug resistance. Infection with K. pneumoniae is reported to induce microtubule collapse and elicit the release of lung endothelial-derived amyloid-beta (A β ) and tau. K. pneumoniae -induced lung endothelial amyloids are heat-stable and transmissible in a prion-like manner. The mechanism(s) of these phenomena are poorly understood, and whether the propagation of the infection-generated proteinopathy is dependent upon tau and/or A β has not been investigated. Hypothesis: Here, we tested the hypothesis that virulent K. pneumoniae infection induces tau dysregulation, microtubule breakdown, increased barrier permeability, and the production and release of cytotoxic amyloids. Methods: CRISPR/Cas9 was utilized to generate tau-/- and APP-/- [APP: amyloid precursor protein (A β )] pulmonary microvascular endothelial cells. Gene deletion was confirmed and monitored via sequencing, RT-qPCR, and immunoblotting. A hypervirulent K. pneumoniae strain was isolated from the

bronchoalveolar lavage of a patient diagnosed with monomicrobial nosocomial pneumonia, deidentified, and characterized (Kp 1-008). Control, tau-/-, and APP-/- cells were infected at a multiplicity of infection of 40:1. Transwell assays were used to assess permeability and bacterial dissemination over time. Amyloids were monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red with birefringence. Infection supernatants were collected, filter-sterilized, boiled and cold shocked to denature non-amyloid proteins, and transferred to naïve wild-type cells to assess independent protein propagation potential. Cytoskeletal dynamics were monitored over time via immunocytochemistry (Anti-tau and anti tubulin antibodies for microtubules; phalloidin to label F-actin). Results: Kp 1-008 induced cytoskeletal disruption, increased permeability, and bacterial dissemination by 3-4 h post-infection. Sterile infection supernatants transferred to naïve cells induced marked barrier disruption, permeability, and cell death within 12 h post-treatment. Interestingly, supernatants from tau-/- and APP- /- cells induced different pathologies, suggesting that either tau or A β alone is sufficient to propagate injury in a strain-dependent manner.

However, maximal transmissible injury required both tau and A β together. Conclusion: K. pneumoniae induces cytoskeletal disruption to promote endothelial barrier permeability and dissemination while generating tau and A β cytotoxins. Moreover, either tau or A β alone are capable of propagating injury from cell-to-cell. However, K. pneumoniae infection-generated tau and A β synergize to facilitate the maximum virulence and potency of the consequent lung endothelial derived proteinopathy.

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Biomedical Research

Cyrill Slezak, PhD; Karaleen Anderson, MD; Tyson Hillock, OMS-II; Mariel Miller, BS; Peter Dungel, PhD; Olga Kopp, PhD; Katja Sterflinger, PhD; Paul Slezak, MD Utah Valley University, Orem, Utah; Ludwig Boltzmann Institute for Traumatology, Austrian Cluster for Tissue Regeneration, University of Natural Resources and Life Sciences, Vienna, Austria 05 SHOCKWAVES INCREASE IN VITRO RESILIENCE OF RHIZOPUS ORYZAE BIOFILM UNDER AMPHOTERICIN B TREATMENT

LFUS, radial pressure waves (RPW), or SW on R. oryzae biofilms. Methods: LFUS biofilms were grown and treated in 96 well plates, whereas all RPW and SW were grown and treated in 24 well plates. SW was also grown and treated in 15mL falcon tubes with a fibrin glue base to mimic tissue biofilm growth. Falcon tube treatment was conducted in a water bath to mimic energy transfer through tissue. LFUS biofilms were treated with a serial dilution of Amp B (16-0.03 μg/mL) in four main conditions: LFUS only, LFUS Amp B, Amp B only, and an untreated control. The LFUS parameters were 550 kHz for 10 min duration. SW was treated with either electrohydraulic (EH) or electromagnetic (EM) applicators. RPW and SW were treated with a predetermined Amp B MIC50 of 8 μg/mL in four main conditions: SW or RPW only, SW or RPW Amp B, Amp B only, and an untreated control. EH parameters were 0.19 mJ/mm2 at 3Hz with 300 pulses. EM parameters were 0.12 mJ/ mm2 at 3Hz with 300 pulses. RPW parameters were 300 pulses with 1 or 3 Barr. EM water bath parameters were 0.55 mJ/mm2 at 3Hz with 300 pulses. XTT and BrdU labeling assays were used to measure cellular respiration and proliferation,

Background: Extracorporeal shockwave (SW) treatment is used to treat chronic soft tissue wounds, including burns and diabetic ulcers. While previous reports suggest that SW are effective at damaging bacterial biofilms, the effect of shockwaves on fungal biofilms has been largely ignored. Fungi have shown a commensal interaction with Staphylococcus aureus , promoting structural and chemical drug resistance. Many studies have shown that low frequency ultrasound (LFUS) treatments disrupt both bacterial and fungal biofilms, specifically with S. aureus . It is therefore likely that LFUS treatment of polymicrobial biofilms would effectively neutralize both fungal and bacterial biofilms and facilitate drug delivery and efficacy. R. oryzae is a leading cause of Mucormycosis and is associated with increased infection in uncontrolled diabetes, trauma, and burn patients. Amphotericin B (Amp B) has been the first line of treatment for Mucormycosis since the 1950s. However, it can have severe side effects including fatal hepato and nephrotoxicity. Despite the growing clinical significance of this disease, novel antifungal susceptibility testing on this species has remained largely uninvestigated. Objective: The aim of this work is to test for improved drug efficacy through synergistic mechanical biofilm disruption when applying

respectively. Confocal and light microscopy with either calcofluor or crystal violet staining observed biofilm structural integrity. Results: LFUS significantly enhanced Amp B effects at concentrations of < 1 μg/mL when compared to Amp B alone (p > 0.0001). Similarly, RPW showed a synergistic effect on Amp B efficacy (p > 0.001). However, both EH and EM increased cellular respiration with Amp B treatment (p > 0.0001 and p > 0.05, respectively). EM treatment in the water bath further showed increased cellular metabolism regardless of receiving Amp B treatment (p > 0.05). Proliferation surprisingly showed a decrease in combined EH and Amp B therapy when compared to Amp B alone (p > 0.0001). Confocal and light microscopy suggested increased biofilm integrity after synergistic treatment. Conclusions: This study provides evidence of LFUS and RPW enhancing the effectiveness of Amphotericin B through synergistic mechanical agitation when treating R. oryzae biofilm. In contrast, the application of SW resulted in a strong biological response of the biofilm, significantly increasing cellular viability, thus lowering the efficacy of Amp B. These findings further support the regenerative potential of SW by illustrating that this effect exists even in fungal biofilms.

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Biomedical Research

1 Alexandra Kuck, BS, OMS-II; 1 Corbin Sapp, BS, OMS-III; 1 Lauren Uram, BS, OMS-III; 2 Emma Kange, BS, OMS-III; 2 Pritty Dwivedy, BS, OMS-IV; 2 Ramu Anandakrishnan, PhD; 2 Robin Varghese, PhD; 3 Aline Andres, RD, PhD; 1 Melissa Lipsmeyer, MS, PhD 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 Edward Via College of Osteopathic Medicine-Virginia, Blacksburg, Virginia; 3 Arkansas Children’s Nutrition Center, Little Rock, Arkansas 06 EXPLORING THE FUNCTION OF THE BACTERIAL-DERVIVED METABOLITE INDOLE-3-PROPIONIC ACID IN PLACENTAL PHYSIOLOGY AND IT’S POTENTIAL PROTECTIVE ROLE AGAINST OBESITY-INDUCED PERTUBATIONS DURING PREGNANCY

Context: Indole-3-propionic acid (IPA) is a bacterial derived metabolite from the gut microbiome that has anti-inflammatory properties and has been shown to decrease oxidative stress in various tissues. With regard to metabolism, IPA is a known regulator of glucose homeostasis and is decreased in the serum of patients with Type 2 diabetes mellitus as well as in patients with obesity. While the molecular functions of IPA are characterized in some tissues, it’s role in female reproductive tissues remains unknown. Objective/Hypothesis: The objective of this study was to determine if placental IPA levels differed in women during the first trimester of pregnancy with overweight/obesity from normal weight counterparts. We also investigated if serum IPA levels were associated with specific dietary patterns as well as markers for glucose homeostasis, inflammation, or lipid metabolism. We furthered our investigation into the protective mechanism of IPA against obesity utilizing human trophoblastic cell lines cultured in adipocyte conditioned media in an in vitro model of obesity. We hypothesized IPA would alter metabolic pathways related to glucose metabolism, inflammation, and oxidative stress. Methods: Serum was collected from n=198

women during the first trimester of pregnancy between 10-12 weeks of gestation. IPA levels were measured by ultra-high performance liquid chromatography-high-resolution accurate mass analysis. Other analytes were measured utilizing various methods. Analyses of IPA associated with specific markers was conducted using simple linear regression models. Characterization of IPA functions in placental trophoblastic cells in our in vitro model of obesity was performed by RNAseq analysis. Results: Serum IPA levels were significantly decreased in women with overweight/obesity (BMI) as well as in women with higher adiposity (%Fat mass). A significant negative correlation was found between serum IPA and %FM. Serum IPA levels were also significantly correlated with dietary fiber intake, HOMA-IR (insulin resistance) and serum insulin. No other associations were significant. RNAseq analysis revealed that IPA significantly altered expression of genes related to inflammation, cellular migration and adhesion, collagen expression as well as other genes implicated in disorders of the placenta in response to an obesogenic environment. Conclusion: Serum IPA levels are diminished in pregnant women with higher adiposity and may reflect a disruption of the microbiome. IPA

levels are significantly associated with serum insulin and lower levels of insulin resistance demonstrating IPA may influence glucose homeostasis and insulin sensitivity during pregnancy. Our RNAseq analysis offers initial insight into the molecular mechanisms of IPA in placental cells. 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 disorders such as gestational diabetes by enhancing the gut microbiome prior to pregnancy.

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Biomedical Research

Erika Lytle, OMS-II 1 ; Rebekah Morrow, PhD 1 ; K. Adam Morrow, PhD 1 ; Chung-Sik Choi, PhD 2 ; Mike Lin, PhD 2 ; Troy Stevens, PhD 2 ; and Sarah Voth, PhD 1 1 Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana; 2 University of South Alabama – Whiddon College of Medicine, Mobile, Alabama 07 EXOY IMPAIRS TAK1-MEDIATED ENDOTHELIAL AMYLOID INNATE DEFENSE

Background: Pseudomonas aeruginosa is a Gram-negative opportunist and the predominant agent responsible for nosocomial pneumonia. Survivors of nosocomial pneumonia suffer from increased rates of disability, morbidity, and mortality post-discharge. Non-virulent P. aeruginosa lung infection elicits endothelial derived cytoprotective amyloid species including broad-spectrum antimicrobials. However, virulent P. aeruginosa utilizes a type III secretion system (T3SS) to inject toxins directly into the cytoplasm of the target cell which inhibits the efficacy of innate endothelial-derived amyloids. T3SS effector exoenzyme Y (ExoY) is a nucleotidyl cyclase found in roughly 90% of clinical strains. In lung capillary endothelial cells, ExoY generates unconstrained cyclic nucleotides cGMP and cUMP. Further, ExoY has been reported to suppress the innate immune response via inhibition of transforming growth factor- β -activating kinase 1(TAK1). Lung microvascular endothelial cells intoxicated with ExoY also release virulent tau cytotoxins that propagate injury and are transmissible among species indicative of a prion strain. Hypothesis: We hypothesize that ExoY intoxication obstructs TAK1 to suppress endothelial amyloid antimicrobicity and promote production of pathogenic tau.

Methods: Control and tau-/- pulmonary microvascular endothelial cells (PMVECs) were infected with isogenic mutants of P. aeruginosa at an MOI of 20:1 both with and without inhibition of TAK1 via potent and selective (5Z)- 7-oxozeanol over a range of concentrations [4 nM, 8 nM (IC50), 16 nM]. Isogenic mutants include ∆PcrV (incompetent T3SS needle), ∆UT (patent needle but genetic deletion of canonical T3SS toxins), ExoY K81M (ExoY incapable of cyclase activity), and ExoY + (expresses and injects ExoY only). Exogenous caged cyclic nucleotide analogs (cGMP-AM and cUMP-AM) will be utilized in the absence of infection to ascertain whether cytosolic increases in these cNMPs alone are sufficient to inhibit TAK1. Amyloids were monitored via immunoblotting with validated antibodies against recombinant controls, thioflavin T fluorescence, and Congo Red birefringence. TAK1, NF- ƙ B, and cytokine levels were measured via immunoblotting and RT-qPCR, respectively. Cytokines and TAK1 activity were measured with ELISA. Amyloid and tau function (antimicrobial vs. pathogenic) will be ascertained through standard antimicrobial and cytotoxicity assays. Results: The release of lung endothelial-derived innate amyloids and cytokines were decreased in direct proportion to the inhibition of TAK1

in a dose-dependent manner. Probing of non virulent infection-derived cell supernatants for antimicrobial amyloid oligomers decreased concordantly with pharmacologic inhibition. Likewise, thioflavin T studies revealed a dose dependent decrease in the release of amyloid species. ExoY intoxication alone inhibited TAK1 activity, transcript, and expression. Moreover, pharmacologic inhibition of TAK1 was sufficient to induce pathogenic tau production and inhibit antimicrobial amyloid release in fidelity with ExoY intoxication. Conclusion: Taken together, these data indicate that T3SS nucleotidyl cyclase effector ExoY inhibits TAK1 to impair host defense and promote a virulent tauopathy secondary to P. aeruginosa infection.

19 2023 Via Research Recognition Day

Biomedical Research

Kristina McLeod-van Amstel, OMS-II; Dr. Rebecca Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana 08 CHARACTERIZING THE ROLE OF AMYLOID BETA IN PULMONARY ENDOTHELIUM AS PART OF THE INFLAMMATORY IMMUNE RESPONSE.

Context: Pseudomonas aeruginosa has been identified as a key infectious organism in the development of pneumonia and sepsis. During infection, the immune system causes pulmonary endothelium to release inflammatory cytokines along with amyloid proteins. certain strains of P. aeruginosa can disrupt the host cell defense and disable the pulmonary endothelium’s autophagy ability, allowing for pathogenic tau and amyloid proteins to accumulate without recycling. Of the amyloid proteins involved, levels of amyloid beta have been shown to increase in concert with the infection as well as decrease upon remission. While tau is known to be cytotoxic to cells and contributory to the pathology of the infection, the role of amyloid beta as it contributes to the inflammatory immune response has yet to be characterized within the pulmonary system. As such, this study aims to understand its role as it contributes to innate host defense within pulmonary health using rat pulmonary vascular endothelial cells and by targeting its precursor protein APP. Objective and/or Hypothesis: The goal of this study is to determine the role of amyloid beta in pulmonary endothelial cells by deleting its precursor protein APP and determining if there is any change in behavior between control and knockout cells in both the normal and

physiological challenge states.

Methods: Pulmonary vascular endothelial cells came from the rat cell line MVR1D. Two clones of cells with CRISPR/Cas9 deleted APP will be compared to the wild type MVR1D and a gRNA cell line that will act as a CRISPR control. The expression of IL-6, TNF, CCL5, and IL-1 β genes in each cell line will be determined using RT-PCR. Cell lines will be cultured in standard medium and assessed for growth through an MTT assay. Later endeavors include infecting the cells with P. aeruginosa and assessing their inflammatory reaction via a cellular cytokine assay. Results/Anticipated Results: The study is currently in progress and results are not yet complete. It is anticipated that the APPKO cell lines will grow at similar rates as the wild type; however, they may lack the robust inflammatory reaction that the wild type would. Conclusion(s): Conclusions not yet reached due to ongoing data analysis.

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Biomedical Research

Naved Salim, BS, OMS-II; Camryn Daidone, BS, OMS-II; Kevin Le, BS, OMS-II; Kristina McLeod van-Amstel, MS, OMS-II; Sarah Voth, PhD; K. Adam Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana 09 ARTIFICIAL INTELLIGENCE IN MICROSCROPIC ANALYSIS

Context: Analysis of microscopic images is a backbone of many areas of research. Digital image processing software such as ImageJ are commonly used to precisely annotate, measure, and analyze images. However, without programming knowledge or Macros, ImageJ is reliant on manual analysis of individual images, a process that is often tedious and leaves data susceptible to human error. Biodock is a new online software that contrasts the traditional manual processing of images as done through ImageJ by allowing users to train Artificial Intelligence (AI) pipelines to identify and quantify objects within an image. Biodock allows users to train pipelines to obtain the desired data and then, once trained, the AI pipelines can be utilized to analyze similar images. Though a potentially less time-intensive and automated approach to image analysis and data collection, the efficacy of Biodock has not been widely tested. Objective: The goal of this study is to determine the efficacy of Biodock AI pipelines in analyzing microscopic images of cells compared to traditional manual analysis by ImageJ. Methods: We tested Biodock’s ability to perform 3 functions compared to ImageJ: measure wound area on scratch wound images,

calculate number of cells on growth curve images, and count number of enclosed loops on network formation images. The sample sizes of images evaluated for scratch wound area, growth curve, and network formation were 126, 45, and 20, respectively. Using ImageJ, wound space area was measured manually using the “freehand selections” function; individual cells and number of enclosed loops within growth curve and network formation images, respectively, were calculated manually using the “multipoint function.” On Biodock these same functions were performed through training three separate AI pipelines, one for each image type, and deploying the trained model to perform these functions on subsequent images. Differences between the means of the data measured from Biodock and ImageJ were calculated using an unpaired two-tailed t-test. We used an alpha value of p = 0.05 to determine statistical significance within a 95% confidence interval. Results: Trained Biodock AI analysis was highly time efficient and resistant to the inherent variability in outcomes that arise via the analyses of an image data set by multiple users. Statistically, there was no significant difference between the Biodock and ImageJ generated image analysis data for scratch wound area,

growth curve, or network formation (p = 0.7024, 0.9517, 0.6800, respectively). Conclusions: These data suggest that Biodock is a promising alternative to ImageJ for quantification of images taken from cell-based functional assays.

21 2023 Via Research Recognition Day

Biomedical Research

Reggie Skains III, MA, OMS-III; Rebekah Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana 10 THE ROLE OF INTRACELLULAR OSTEOPONTIN IN PULMONARY ARTERIAL HYPERTENSION

Context: This research is currently being done to further understand the pathogenesis of pulmonary arterial hypertension (PAH). There is a proposed theory that the endothelial to mesenchymal transition of pulmonary artery endothelial cells plays a role. This project will examine whether the loss of intracellular osteopontin enhances endothelial dysfunction in pulmonary arterial endothelial cells in pulmonary arterial hypertension. PAH is currently treated only by addressing the symptoms and not the underlying pathophysiology. With better understanding of the pathogenesis and role of different inflammatory molecules, there is hope this disease can be treated at the source of disease instead of just managing the symptoms associated with it. Objective: The objective is to examine whether loss of intracellular osteopontin enhances endothelial dysfunction in pulmonary artery endothelial cells in PAH. Methods: Gel electrophoresis, western blot, ELISA Results: There was variable expression of selected smooth muscle and fibroblast markers in the control wild type cell lines and infected cell lines. Control state and disease state cell

lines did not express markers in expected ways. This can be due to technical error or unexpected expression from the cell lines. This area of research and specific project is currently the focus of the Morrow lab, led by Drs. Adam and Rebekah Morrow and is still ongoing. Future research can focus on other smooth muscle and endothelial markers, such as vimentin, fibronectin, and alpha smooth muscle actin to see if they play a role in the endothelial to mesenchymal transition. Conclusions: While the results of this specific research project are not conclusive of the expression of osteopontin in the diseased and control cell lineages, whether this is due to technical error or not, future research should be continued to pinpoint the role of OPN in PAH.

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