2022 IEBMC Program booklet by LeeFerg

IEBMC 8th annual

INTERNATIONAL EXPERIMENTAL BIOLOGY & MEDICINE CONFERENCE

APRIL 29—MAY 1, 2022 CENTRAL STATION HOTEL, MEMPHIS, TN

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WELCOME Dear IEBMC Attendees, I want to welcome you to Memphis, Tennessee, “Home of the Blues & Birthplace of Rock ‘n’ Roll,” for our 8th Annual International Experimental Biology and Medicine Conference (IEBMC). We created IEBMC in 2013 for the purpose of creating a platform for bringing together interdisciplinary researchers in the fields of experimental biology that have great impact on the practice of medicine. To create dynamic meetings where new ideas and collaborations can unfold, we limit our attendance to 200-300 scientists working in the selected area. This year the program committee has selected the important field of regenerative medicine and has put together a stellar group of plenary speakers with a keynote lecture by Dr. Arnie Caplan, “the father of mesenchymal stem cells.” We are also excited to offer you our Beale Street Evening Memphis music event featuring the Sensational Barnes Brothers and the Stax Music Academy Alumni Band, 926. On behalf of our sponsors: The Society of Experimental Biology and Medicine (SEBM), Experimental Biology and Medicine, the University of Tennessee Health Science Center (UTHSC), Tennessee Institute of Regenerative Medicine (TennIRM), the University of Memphis (UofM), Medtronic, Revotek, and the Regenerative Medicine Foundation, I hope you enjoy IEBMC and the city of Memphis.

Steve Goodman, PhD Vice Chancellor for Research The University of Tennessee Health Science Center Editor-in-Chief, Experimental Biology and Medicine President and CEO, Clinical Trials Network of Tennessee (CTN2)

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ORGANIZERS AND CONTRIBUTORS This year's conference is sponsored by the Society for Experimental Biology and Medicine and its journal, Experimental Biology and Medicine. It is hosted by the University of Tennessee Health Science Center and the Tennessee Institute of Regenerative Medicine, and supported by Revotek, Medtronic, the Regenerative Medicine Foundation, and the University of Memphis.

ORGANIZING COMMITTEE Steve R. Goodman, PhD, Chair Vice Chancellor for Research, University of Tennessee Health Science Center Editor-in-Chief, Experimental Biology and Medicine President and CEO, Clinical Trials Network of Tennessee (CTN2) Jian Feng, PhD State University of New York at Buffalo Lee Ferguson Director of Communications and Marketing, Office of Research, University of Tennessee Health Science Center Greg Harris Senior Director of Development, University of Tennessee Foundation Jessica Homa Executive Director, Society of Experimental Biology and Medicine

CONTRIBUTORS

Y. James Kang, PhD University of Tennessee Health Science Center Chief Scientist, Tennessee Institute of Regenerative Medicine Roshan Kumari SEBM Student Member, University of Tennessee Health Science Center Agnes Luo, PhD University of Cincinnati College of Medicine Councilor, Society of Experimental Biology and Medicine Willliam Slikker, PhD Director, National Center for Toxicological Research/FDA Tom Thompson, PhD University of Cincinnati College of Medicine President, Society of Experimental Biology and Medicine John Wikswo, PhD Director, Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University Warren Zimmer, PhD Texas A&M Health Science Center Membership Committee Co-Chair and Nominating Committee Chair, Society of Experimental Biology and Medicine

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TABLE OF CONTENTS SPEAKERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 SCHEDULE AT-A-GLANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 PETER STAMBROOK TRIBUTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ABSTRACTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

COVID-19 PROTOCOL All attendees of the 2022 IEBMC must be fully vaccinated against COVID-19. Individuals are considered fully vaccinated two weeks after receiving the final dose required by the manufacturer. Attendees will need to present a facsimile of a U.S. Center for Disease Control inoculation record, or similar verifiable document issued by a U.S. federal agency or an official state agency in another country, when picking up your meeting materials at the session check-in tables (Grand Hall entrance).

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KEYNOTE SPEAKER Arnold I. Caplan, PhD Director of the Skeletal Research Center Professor of Biology Case Western Reserve University “MSCs can be curative for diseases, including COVID-19”

PLENARY SESSION SPEAKERS Yoko M. Ambrosini, DVM, MPVM, PhD, DACVIM (SAIM) Assistant Professor, Department of Veterinary Clinical Sciences College of Veterinary Medicine Washington State University, PhD "Pathomimetic human intestinal disease-on-a-chip: From organomimicry to precision medicine”

Jian Feng, PhD Professor, Department of Physiology and Biophysics Jacob School of Medicine and Biomedical Sciences State University of New York at Buffalo “Generation of mouse-human chimeric embryos”

Daniel Garry, MD, PhD Abbott Medical Endowed Chair in Cardiovascular Sciences Director, Regenerative Medicine and Sciences Program Director, Paul and Sheila Wellstone Muscular Dystrophy Center University of Minnesota “Interspecies chimeras as a platform for xenotransplantation”

W. Keith Jones Director, Pharmacovigilance Professor, Loyola University Chicago Stritch School of Medicine “The Molecular Basis of Exosomal microRNA Packaging and Action in Myocardial Protection”

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Y. James Kang, PhD Chief Scientist, Tennessee Institute of Regenerative Medicine (TennIRM) Professor, College of Graduate Health Sciences University of Tennessee Health Science Center “Reactivation of HIF-1 regulated angiogenesis promotes myocardial regeneration”

Agnes Luo, PhD Associate Professor of Molecular Genetics University of Cincinnati “Modulating endogenous repair mechanisms to enhance functional recovery after CNS injuries”

Peter Loskill, PhD Professor for Organ-on-Chip-Research Eberhard Karls University Tübingen and the Natural and Medical Sciences Institute (NMI) “Microphysiological platforms, organoids and enabling technologies: Integrated Organ-on-Chip models recapitulating complex human tissues”

Keith March, MD, PhD, FACC Director, UF Center for Regenerative Medicine Professor of Medicine, UF Division of Cardiology University of Florida “Next-generation Cell-based Therapies: Cellular Therapies in a Bottle, on the Shelf, and treating Organs”

Megan McCain, PhD Assistant Professor, Department of Biomedical Engineering Viterbi School of Engineering University of Southern California “Engineering Cardiac and Skeletal Muscle on a Chip Systems for Multi-Modal Physiological Analysis and Disease Modeling"

Shannon McKinney-Freeman, PhD Associate Member, Department of Hematology St. Jude Children’s Hospital “Uncovering novel regulation of hematopoietic stem cells during homeostasis and disease”

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Guoli Ming, MD, PhD Perelman Professor of Neuroscience Perelman School of Medicine University of Pennsylvania “Modeling human brain development and disorders using brain organoids”

Munemasa Mori, MD, PhD Assistant Professor of Medicine, Columbia Center for Human Development Pulmonary Allergy and Critical Care Medicine, Department of Medicine Columbia University Irving Medical Center “Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells”

Cindi Morshead, PhD Professor and Chair, Division of Anatomy University of Toronto “Activating endogenous neural precursor cells to promote neural repair”

Hannele Ruohola-Baker Professor of Biochemistry Associate Director, Institute for Stem Cell and Regenerative Medicine (ISCRM) College of Medicine University of Washington “Decoding regeneration using computer designed proteins”

Antonio Santos, PhD Post-Doctoral Fellow (Calvin Kuo’s Laboratory) Stanford University “A human autoimmune organoid model of celiac disease”

Robert J. Schwartz, PhD Hugh Roy and Lillian Cranz Cullen Distinguished University Professor Department of Biology & Biochemistry University of Houston “SRF Mutants Named Stemins Induce a Cardiac Myocyte Cell Regenerative Program”

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Kyung Sung, PhD FDA Office of Tissues and Advanced Therapies, Division of Cellular and Gene Therapies, Cellular and Tissue Therapy Branch “Regulation of Regenerative Medicine cellular products and research to support the development of these products”

Gábor Tigyi, MD, PhD, DSc Harriet Van Vleet Endowment Professor, Department of Physiology Executive Director, Tennessee Institute of Regenerative Medicine (TennIRM) University of Tennessee Health Science Center “Drug Development Strategies to Rescue Intestinal Stem Cells and Organoids from Radiation-induced Genotoxic Stress”

John P. Wikswo, PhD Gordon A. Cain University Professor A.B. Learned Professor of Living State Physics Vanderbilt Institute of Integrative Biosystems Research and Education Vanderbilt University “Closing the hermeneutic circle of biology: systems biology, organs-on-chips, mass spectrometry, and synthetic biology” 7

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PROGRAM FRIDAY, APRIL 29, 2022 8:30am - 9:00am

WELCOME CEREMONY

9:00am - 10:00am

Keynote Speaker Arnold I. Caplan, Case Western Reserve University "MSCs can be curative for diseases, including COVID-19"

PLENARY SESSION 1: STEM CELL BIOLOGY Chair: Jian Feng Co-Chair: Shannon McKinney-Freeman

10:00am - 10:35am

Jian Feng, State University of New York at Buffalo "Generation of mouse-human chimeric embryos"

10:35am - 11:10am

Daniel Garry, University of Minnesota "Interspecies chimeras as a platform for xenotransplantation"

11:10am - 11:20am

BREAK

11:20am - 11:55am

Hannele Ruohola-Baker, University of Washington "Decoding regeneration using computer designed proteins"

11:55am - 12:30pm

Shannon McKinney Freeman, St. Jude Children’s Research Hospital "Uncovering novel regulation of hematopoietic stem cells during homeostasis and disease"

12:30pm - 1:30pm

LUNCH

DAY 1 PLENARY SESSION 2: ORGANOIDS Chair: Tom Thompson Co-Chair: Gábor Tigyi 1:30pm - 2:05pm

2:05pm - 2:40pm

Antonio Santos, Stanford University "A human autoimmune organoid model of celiac disease" Munemasa Mori, Columbia University "Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells"

2:40pm - 2:50pm

BREAK

2:50m - 3:30pm

Gábor Tigyi, University of Tennessee Health Science Center "Drug Development Strategies to Rescue Intestinal Stem Cells and Organoids from Radiation-induced Genotoxic Stress"

3:30pm - 4:15pm

Presentation on SEBM and EBM Steve Goodman, University of Tennessee Health Science Center Tom Thompson, University of Cincinnati

4:15pm - 6:00pm

BREAK

6:00pm - 7:30pm

POSTER SESSIONS

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PROGRAM SATURDAY, APRIL 30, 2022 8:30am - 8:45am

PETER STAMBROOK AWARD CEREMONY Tom Thompson, Steve Goodman, Wayne Zimmer

PLENARY SESSION 3: ORGANS ON A CHIP Chair: John Wikswo Co-Chair: Steve Goodman 8:45am - 9:20am

John Wikswo, Vanderbilt University "Closing the hermeneutic circle of biology: systems biology, organs-on-chips, mass spectrometry, and synthetic biology"

9:20am - 9:55am

Megan McCain, University of Southern California "Engineering Cardiac and Skeletal Muscle on a Chip Systems for Multi-Modal Physiological Analysis and Disease Modeling"

9:55am - 10:05am

BREAK

10:05am - 10:40am

Yoko Ambrosini, Washington State University "Pathomimetic human intestinal disease-on-a-chip: From organomimicry to precision medicine"

10:40am - 11:15am

Peter Loskill, Eberhard Karls University Tübingen "Microphysiological platforms, organoids and enabling technologies: Integrated Organ-on-Chip models recapitulating complex human tissues"

ABSTRACT PRESENTATIONS Chair: Warren Zimmer 11:15am - 11:30am

Xiang Li, SUNY Upstate Medical University "ABI1 regulates STAT3 transcriptional activity through a DNA binding mechanism"

11:30am - 11:45am

Chao Ma, New York University "Leukemia-on-a-Chip for Modeling and Decoding Chemotherapy Resistance"

11:45am - 12:00pm

Solomon Owumi, University of Ibadan "Unfettered antibiotics access, microbial resistance and carcinogenesis"

12:00pm - 12:15pm

Roshan Kumari, University of Tennessee Health Science Center "Role of SMAD2/3 on Diet-Induced Obesity and Adiposity"

DAY 2 12:15pm - 1:15pm

LUNCH

PLENARY SESSION 4: REGENERATIVE MEDICINE AND THE CARDIOVASCULAR SYSTEM Chair: Y. James Kang Co-Chair: Robert J. Schwartz 1:15pm - 1:50pm

Robert J. Schwartz, University of Houston "SRF Mutants Named Stemins Induce a Cardiac Myocyte Cell Regenerative Program"

1:50pm - 2:25pm

W. Keith Jones, Loyola University Chicago "The Molecular Basis of Exosomal microRNA Packaging and Action in Myocardial Protection"

2:25pm - 2:35pm

BREAK

2:35pm - 3:10pm

Y. James Kang, University of Tennessee Health Science Center "Reactivation of HIF-1 regulated angiogenesis promotes myocardial regeneration"

3:10pm - 3:45pm

Keith March, University of Florida "Next-generation Cell-based Therapies: Cellular Therapies in a Bottle, on the Shelf, and treating Organs"

3:45pm - 4:30pm

David Boothman Awards and SEBM/EBM/IEBMC Awards

4:30pm - 6:00pm

BREAK

6:00pm - 8:30pm

BEALE STREET THEMED PARTY featuring the Stax Music Academy Alumni Ensemble and the Sensational Barnes Brothers 12

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PROGRAM DAY 3 SUNDAY, MAY 1, 2022 PLENARY SESSION 5: REGENERATIVE MEDICINE AND THE NERVOUS SYSTEM Chair: Agnes Luo Co-Chair: Y. James Kang 8:30am - 9:05am

Agnes Luo, Cincinnatti University "Modulating endogenous repair mechanisms to enhance functional recovery after CNS injuries"

9:05am - 9:40am

Guoli Ming, University of Pennsylvania "Modeling human brain development and disorders using brain organoids"

9:40am - 9:50am

BREAK

9:50am - 10:30am

Cindi Morshead, University of Toronto "Activating endogenous neural precursor cells to promote neural repair"

FINAL LECTURE Chair: Bill Slikker, Jr. 10:30am - 11:00am

Kyung Sung, FDA Office of Tissues and Advanced Therapies "Regulation of Regenerative Medicine cellular products and research to support the development of these products"

11:00am - 11:30am

Closing Remarks Steve Goodman, Tom Thompson

12:15pm - 1:15pm

FAREWELL LUNCH, for speakers and SEBM/EBM leadership

TRIBUTE TO PETER STAMBROOK This year, we remember and honor the life of Peter Stambrook, PhD, past SEBM president and 2015 recipient of the SEBM Distinguished Scientist Award, who passed away in 2020. He was 79. Dr. Stambrook was an internationally respected cancer researcher whom friends and colleagues describe as a brilliant scientist, visionary researcher, and dedicated educator who tirelessly supported students. Born in London, Dr. Stambrook immigrated to the United States with his family in the early 1950s. After receiving his PhD from the State University of New York at Buffalo, he began his career in the Department of Embryology at the Carnegie Institution of Washington in Baltimore and eventually joined the faculty at Case Western Reserve University, where his lab was the first in Cleveland to use recombinant DNA technology. He moved to the University of Cincinnati, where he remained a faculty member for 39 years. During his tenure, he served as Francis Brunning Professor and Chair of the Department of Cell and Cancer Biology, and was eventually named Distinguished Research Professor. Over the course of his career, Dr. Stambrook attracted millions of dollars in research funding and made seminal discoveries in his field. He was a leader in the field of DNA replication and cell cycle regulation. His scope of interest extended to cancer cells and embryonic stem cells and, in later years, to gene mutation and genomic instability as they relate to cancer. Most recently, Dr. Stambrook focused on a critical signaling pathway that responds to DNA damage and subsequent cell cycle regulation. Dr. Stambrook received numerous honors in his lifetime, including a Senior Fogarty Fellowship at St. Mary’s Hospital Medical School in London, the George Rieveschl Award for Distinguished Scientific Research, election as a Fellow of the Graduate School of the University of Cincinnati, election as a Fellow of the American Association for the Advancement of Science, and election as an Overseas Fellow of the Royal Society of Medicine. He was the 2013 recipient of the Drake Medal from the University of Cincinnati and the recipient of the Environmental Mutagenesis and Genomics Society award. Among his many activities, Dr. Stambrook served as the Scientific Director and Chair of the International Scientific Council for the Israel Cancer Research Fund. He has also served on the board of the American Cancer Society, Ohio Division, and as Past President of the Environmental Mutagenesis and Genomics Society. He was Editor-in Chief of Mutation Research, Associate Editor of Experimental Biology and Medicine, a member of the Advisory Board for the Biology and Biotechnology Research Program at the Lawrence Livermore National Laboratory, and served on the Board of Directors of the Public Affairs Executive Committee of FASEB. The SEBM and scientific community offers its condolences to his family and many friends.

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PARTY DON'T MISS SATURDAY NIGHT'S BEALE STREET THEMED

THE SENSATIONAL BARNES BROTHERS

THE STAX MUSIC ACADEMY ALUMNI BAND, 926

Saturday, April 30, 2022 6:00pm – 9:00pm Central Station Hotel Grand Ballroom Saturday night's Beale Street themed party will feature the Sensational Barnes Brothers and the Stax Music Academy Alumni Band, 926. Made possible by a generous donation from Revotek.

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ABSTRACTS *Denotes Young Investigator Award winner

ORAL PRESENTATION

Role of SMAD2/3 on Diet-Induced Obesity and Adiposity* Roshan Kumari1,2, Tahliyah S. Mims1,4, Michelle A. Puchowicz1, Joseph F. Pierre1,4, and Chester W. Brown1,2,3 1Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN; 2Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, TN; 3Le Bonheur Children’s Hospital, Memphis, TN; 4Department of Nutritional Sciences, College of Agriculture and Life Science, UW-Madison

Objective: Obesity is major medical problems worldwide. SMAD2/3 proteins, the downstream mediators of activin signaling, regulate many biological processes including maintaining body fat composition. We previously determined individual role of Smad2 and Smad3 on diet-induced obesity (DIO) and adiposity. However, the combined adipose tissue specific requirements of Smad2 and Smad3 remained unknown. Here, we sought to determine the combinatorial requirement of Smad2/3 on diet-induced obesity, adiposity, and metabolism. Research Design: To assess the adipose-selective combined requirements of Smad2/Smad3, we generated Smad2/3 conditional knockout mice (Smad2/3cKO) by crossing Smad2/3 “floxed” mice with Adiponectin-Cre mice. Smad2/3cKO and control mice (Smad2/3f/f) were fed low-fat diet (LFD) and high- fat diet (HFD) and examined adipose tissue specific phenotype. Results: Adipose selective combined reduction of Smad2/3, Smad2/3cKO mice displayed sex selective phenotype with no change in body weight on LFD or HFD in male while female mice were protected from DIO with reduction in fat mass in both visceral and subcutaneous depot. To understand the underlying molecular mechanism that contributed to the observed phenotype, we looked at metabolic consequences of Smad2/3 reduction. Smad2/3cKO mice showed higher oxygen consumption and energy expenditure indicative of higher metabolic rate. This was corroborated with the downregulation of several key genes involved in lipogenesis and lipolysis in subcutaneous depot. Conclusions: Our findings suggest that the combined loss of SMAD2/3 in adipose tissue display sex selective phenotype with prominent roles in females with protection from diet induced obesity. These results support the rationale to understand and target SMAD-mediated pathways to treat obesity.

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ORAL PRESENTATION

Leukemia-on-a-Chip for Modeling and Decoding Chemotherapy Resistance* Chao Ma, Weiqiang Chen Department of Mechanical and Aerospace Engineering, New York University, Brooklyn, NY USA; Department of Biomedical Engineering, New York University, Brooklyn, NY USA; Perlmutter Cancer Center, NYU Langone Health, New York, NY

B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer among children and characterized by overproduction of B-cell blasts within bone marrow (BM). The ability to study the interaction among B-ALL and BM is limited by current in vivo experimental methods. Here we report a novel 3D microfluidics-based organotypic ‘Leukemiaon-a- Chip’ comprised a vascularized central sinus region and a medullary cavity serves as an interface of leukemia and niche interactions, to dissect the underlying mechanisms. The system was characterized by time-lapse imaging, single-cell mRNA sequencing (scRNA- Seq), fluorescence immunostaining, and drug testing. We first mapped the heterogeneity of distinct human B-ALL types, such as favorable ETV-RUNX+ and unfavorable Ph+, with scRNA-Seq and found that B-ALL in both niches were activated in TNFA signaling via NF-ҡB. Additionally, we found that BM sub-niches (perivascular and endosteal) differentially regulated leukemia progression and dormancy, highlighting spatial heterogeneity. Finally, we demonstrated the preclinical use of our platform to screen niche- co-targeting regimens. We believe this model presents a powerful tool for drug screening in a more physiologically relevant context.

P3 ORAL PRESENTATION

Unfettered antibiotics access, microbial resistance and carcinogenesis* Solomon E. Owumi1, Eseroghene S. Najophe1, Moses Otunla1, Adegboyega A. Oyelere2 1Department of Biochemistry, University of Ibadan, Ibadan, Nigeria; 2School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

Antibiotics are freely accessible over the counter (OTC) with limited regulation in most Nigerian pharmacies. The tendency of multi-strain microbial resistance potentially exists due to unfettered antibiotic dispensation. Aflatoxin B1 (AfB1) is a potent hepatocarcinogen prevalent in the tropics, contaminates consumed grains in Nigeria. Unfettered OTC antibiotics access, AfB1- contaminated grains coupled with disparate access to food-derived phytochemicals in diet may portend differential predisposition to carcinogenesis in an endemic population. Here we present preliminary results showing that AfB1 significantly (p<0.05) increased pro- carcinogenic inflammatory biomarkers (NO, TNF-α, IL-1β levels and MPO activity) and inhibited IL-10 in rats hepatorenal system. Besides, AfB1 increased(p<0.05) oxidative stress exemplified by decreased antioxidant enzymes (SOD, CAT, GPx, and GST), increased reactive oxygen and nitrogen species (RONS), raised lipid peroxidation and apoptotic caspase-3 activity. Similarly, reduction (p<0.05) in cellular GSH were detected in treated rats. These observed alterations were reversed by co-treatment with the phytochemical Gallic Acid. Furthermore, we will investigate the effects of rats' exposure to selected antibiotics– Azithromycin, Clarithromycin, Ampicillin, and Ciprofloxacin – on AfB1, N-methyl-N- Nitrosourea (NMU) and 1,2-dimethylhydrazine (DMH) carcinogenesis to comprehend the relationship amid reduced gut microflora and carcinogenesis. Our findings may illuminate the roles of microflora in carcinogenesis associated with specific carcinogens AfB1 and DEN (Liver cancer), NMU (Breast cancer) and DHM (Colon cancer) target organs. Keywords: Aflatoxin B1, phytochemicals, oxido-inflammation, carcinogenesis: antibiotics- microbes, liver, breast and colon, AfB1, NMU and DMH.

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ORAL PRESENTATION

ABI1 regulates STAT3 transcriptional activity through a DNA binding mechanism* Xiang Li1,2, Baylee A. Porter1,2, Allysa P. Kemraj1, Alaji Bah1, and Leszek Kotula1,2 1Department of Urology, SUNY Upstate Medical University, Syracuse, NY; 2Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY

Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa as evidenced by its loss or downregulation in high grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial- mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. Cell Commun Signal. 2019). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggested the possibility that it plays a role in EMT through directly regulating transcriptional activity by DNA binding. To examine this hypothesis we set out to analyze ABI1-DNA binding. We purified ABI1 HHR and demonstrated its in vitro interactions with different homeobox DNA-binding consensus of double- stranded DNA sequences. We found that the presence of alternatively spliced Exon 4-encoded sequence, located in the C-terminus of HHR, regulates binding of ABI1 to DNA. We also established that the interaction of the full-length ABI1 and STAT3 occurs through protein phase separation and ABI1/DNA binding mechanisms. Structural NMR studies confirmed ABI1 binding to DNA. Subsequent functional studies using DU145 CRISPR KO cell lines expressing wild type or HHR mutants of ABI1 demonstrated that HHR regulates the nuclear localization and transcriptional activity of STAT3. We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression.

Utilizing Yeast As Model Organism For Drug Discovery Against Eukaryotic Infections* Raphael Lartey Abban1,2,3, Ethel Juliet Blessie1,2,3, Patrick K. Arthur1,2,3 1Department of Biochemistry, Cell and Molecular Biology; 2West Africa Centre Cell Biology of Infectious Disease; 3University of Ghana, Legon

Eukaryotic pathogens are responsible for several serious human diseases, however, most drugs used as treatment regimens are ineffective while others still in use are toxic. This study seeks to identify bioactive molecules from wood decay fungal (WDF) extracts with activity against eukaryotic pathogens (especially Candida albicans and Plasmodium

falciparum). The Saccharomyces cerevisiae model organism for eukaryotic cell biology studies is extensively used to screen bioactive molecules because it allows for rapid. Twelve WDF cultures were extracted with ethyl acetate, and the extracts were screened against C. albicans and S. cerevisiae by the disc diffusion method. Phenotypic array assays were also conducted on selected wood decay fungal extracts. The 3 most active extracts were then fractionated via bioactivity guided fractionation using different chromatographic techniques (solvent partition chromatography, size-exclusion chromatography and preparative TLC). The antifungal and antiplasmodial activities of the fractions were confirmed using MTT assay and SYBR Green 1 growth inhibition assays respectively. The phenotypic array assays performed on the selected wood decay fungal extracts showed different responses to C. albicans and S. cerevisiae, and this led to the selection of top the 3 most-effective extracts as the best candidate for natural product isolation. After an extensive bioactivity-guided fractionation, 49 out of a total of 125 2D Preparative TLC fractions showed antifungal activities in the MTT assay while 47 of the 55 second dimensional preparative TLC fractions showed antiplasmodial activities in the SYBR Green 1 growth inhibition assay. These findings define wood decay fungi as a viable source of antifungal and antiplasmodial compounds, which can be pre-selected using S. cerevisiae over hemolysis problems posed by the total fungal extracts. It will take larger volumes of fungal cultures to finally isolate the many active compounds for structural development and possible clinical development.

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Moringa oleifera extract extenuates Echis ocellatus venominduced toxicities, histopathological impairments and inflammation via enhancement of Nrf2 expression in rats Adeyi Akindele Oluwatosin1*, Adeyemi Sodiq Opeyemi1, Effiong, Eno-Obong Priscilla1, Ajisebiola Babafemi Siji2, Adeyi Olubisi Esther3 and James Adewale Segun3 1Animal Physiology Unit, Department of Zoology, University of Ibadan, Nigeria; 2Department of Zoology, Osun State University, Oshogbo, Nigeria; 3Department of Biochemistry, Federal University of Agriculture, Abeokuta, Nigeria; 4Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria *Corresponding Author:delegenius@yahoo.com

Echis ocellatus envenoming is known to be responsible for more snakebite fatalities than all other African snake species combined. Studies have established that Moringa oleifera possesses attenuating effects against systemic toxicities caused by snake venoms. However, investigations on its effectiveness against envenoming of E. ocellatus in vivo has not been established. Thirty male rats were grouped into six groups (n=5). Group 1 served as Normal control, groups 2 to 6 were envenomed with a single intraperitoneal injection of 0.22 mg/kg (LD50) of E. ocellatus venom. Group 2 was untreated while group 3 to 6 were treated post-envenoming with 0.2 mL of polyvalent antivenom, 200, 400 and 600 mg/kg of M. oleifera ethanol extract respectively. M. oleifera extract significantly (p<0.05) normalized the altered haematological indices and blood electrolytes profiles. Extract attenuated venom-induced cellular dysfunctions, with a significant increase in nuclear factor erythroid-2-related factor 2, and concomitant downregulation of increased antioxidant enzymes (SOD and CAT) activities in serum and heart of envenomed treated rats. M. oleifera extract normalized the elevated tumour necrosis factor-α and interleukin-1β in serum and heart tissues of treated rats. Further, IgG titre value was significantly (p<0.5) higher in envenomed untreated group compared to treated groups. Hemorrhagic, hemolytic and coagulant activities of the venom were strongly inhibited by the extract in a dose-dependent manner. Lesions noticed on tissues of vital organs of untreated rats were abolished by M. oleifera extract. Our findings substantiate the effectiveness of M. oleifera as antivenom agent against E. ocellatus envenoming. Keywords: Moringa oleifera, Anti-venom, Echis ocellatus, Inflammation

Defining TCF4's role as a novel key regulator in mediating neuroblastoma cell identity Nour Aljouda, Dewan Shrestha, Megan Walker, Satyanarayana Alleboina, Yong Cheng, Kevin Freeman The University of Tennessee Health Science Center, Memphis, TN

Neuroblastomas (NB) are embryonal childhood tumors that derive from the multipotent neural crest cells (NCCs) of the peripheral nervous system. NB accounts for more than 15% of all childhood cancer-related deaths. Despite the most intensive multimodal therapy, more than 50% of patients with high-risk NB relapse with often fatal, resistant disease. Finding novel treatments, especially for relapse disease, is desperately needed for high-risk NB. In cancers, distinct transcription factors TFs networks forming core regulatory circuitries (CRCs) control gene expression programs that drive cell identity. Recent studies reported the presence of two types of identity states in NB tumors: one establishing a more proliferative adrenergic (ADRN) cell state and a second establishing a more invasive, therapy-resistant mesenchymal (MES) cell state. While most studies are investigating the CRC members for targeting MYCN amplified NB (ADRN) or the MES/NCCs cell states, most primary tumors are heterogeneous, comprised of cells with both identities. Thus, our work is focused on identifying universal factors shared across different lineage states to determine whether targeting both identities could be a valuable strategy for NB treatment. NB was identified from a screen of 673 cancer cell lines as highly sensitive to the BET-inhibitor JQ1. In our work, we identified the class I basic helix-loop-helix transcription factor (bHLH) TCF4 as a critical target of JQ1 mediated cell death in NB. TCF4 functions as a transcriptional hub that heterodimerizes with class II (bHLH) TFs including the proneural gene ASCL1 (ADRN), and TWIST1 a master regulator of the MES state. Heterodimers formed between TCF4 and ASCL1, and TWIST1, have been demonstrated to provide lineage-specific differentiation from embryonic stem cells. Here, we hypothesize that TCF4 is a cell dependency gene in NB that is crucial for determining NB identity. Using RNA seq analysis of TCF4 dox-inducible shRNA stable cell lines, we found that the gene expression changes in TCF4-depleted cells indicate a role for TCF4 in differentiation, EMT, cell signaling, and neurodevelopment. Interestingly, gene set enrichment analysis (GSEA) indicates that both identity states are suppressed after silencing of TCF4. Our data also shows that inducible decrease of TCF4 protein resulted in a significant inhibition of NB cell growth, induction of apoptosis, and impaired tumor growth in vivo. Furthermore, we identified TCF4 targets in NB by combined analysis of TCF4 ChIP-seq data and gene expression changes following TCF4 knockdown. ENRICHR analysis suggests that differentially expressed genes in TCF4-knockdown cells that also have a TCF4 ChIP-seq peak are targets of the TFs TWIST1 (MES) and HAND1 (ADRN) that interact directly with TCF4. In conclusion, our preliminary data supports the hypothesis that TCF4 is a master transcriptional regulator of NB oncogenic program that is crucial for maintaining NB identity states.

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REGENERATIVE MEDICINE

A comparison of glycemic control and Lipid profile among patients of diabetic dyslipidemia and type 2 diabetes mellitus in Pakistani population* Maria Arif*, Asifa Majeed* *Department of Biochemistry and Molecular Biology, Army Medical College, National University of Medical Sciences, Rawalpindi

Background: Diabetes Mellitus is a metabolic syndrome, which is characterized by hyperglycemia due to relative or absolute decrease in insulin. It accompanies several metabolic complications, poor production of insulin and clearance of lipoprotein. Dyslipidemia is one of these complications of type 2 diabetes mellitus, it is linked with atherosclerosis. Deranged lipid profile is caused by insulin resistance. The importance of glycemic control in patients with T2DM to reduce the risk of micro-vascular and macro-vascular complications is well established and widely recognized by current clinical guidelines, as American Diabetes Association (ADA) and European Association for the Study of Diabetes (EASD). Poor glycemic control and hypertension are the predictors of dyslipidemia in type 2 diabetes mellitus. In T2DM, exchange of cholesterol between LDL and HDL by the help of cholesterol ester transfer protein is increased. Material & Methods: The study was conducted at multi-disciplinary lab, Army Medical College, National University of Medical Sciences, Rawalpindi, after approval of ethical review committee. It was a cross-sectional comparative study. The study technique was non-probability sampling. Duration of the study was two years. Total 300 subjects were divided in three groups; each group contains 100 subjects. World Health Organization (WHO) diagnostic criteria were used for diagnosis of patients. Blood lipid profile was quantified by measuring absorbance in spectrophotometer. The present study is based on exploring the effects of dyslipidemia in patients of type 2 diabetes mellitus as compare to normal healthy controls. Results: The ANOVA test was applied for comparing means. Mean ± SD value between the groups has been find out by the help of Post Hoc Tukey test Lipid profile was found to be statistically significant between the three groups. Conclusion: BSF, HbA1c, lipid profile and BMI were found to be statistically significant among the three groups. It was also seen that BSF, HbA1c and lipid profile were also statistically significant among diabetics and dyslipidemia patients as compare to normal healthy controls. Conclusion: BSF, HbA1c and Lipid profile was found to be linked with diabetic dyslipidemia in addition to T2DM in Pakistani population.

In Vitro Evaluation of the Osteoinductive Potential of Chitosan Membranes Loaded with Raspberry Ketone* Matthew Atwill, Joel D. Bumgardner Biomedical Engineering Department, University of Memphis and University of Tennessee Health Science Center Joint Program, Memphis, TN

Guided bone regeneration (GBR) membranes are used to guide healing in oral bone defects by preventing soft tissue invasion of regenerating bone sites. Electrospun chitosan membranes (ESCM) have exhibited potential for use in GBR applications.1 ESCM also have potential for local delivery of therapeutics for enhancing healing due to high surface area of the nano-fibers1. Raspberry ketone (RK) is a compound found in berries that has exhibited osteogenic potential in

vitro2. This work evaluated the effect of RK released from ESCM on growth, alkaline phosphatase (ALP) expression, and calcium-phosphate deposition of W-20-17 mouse marrow cells over 21 days in culture. Experiments were conducted using osteogenic medium with or without 25ng/mL BMP-2 to evaluate interactions between released RK and BMP-2. After 14 days, cells in positive controls and RK ESCM groups began to differentiate as evidenced by increase in ALP as compared to negative controls. The decreased number of cells at day 21 for positive control and RK ESCM group corresponded to increased mineralization. Negative controls did not mineralize and continued to proliferate. However, lack of difference in ALP and Ca deposition between the BMP-2 control and RK ESCM groups may indicate RK had minimal effect on osteodifferentation. However, RK was rapidly released from ESCM and removed from culture, while BMP-2 was maintained at constant levels in culture. A more sustained RK release pattern would likely yield more realistic differentiation.

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REGENERATIVE MEDICINE

Evaluation of neuronal repair process on focal demyelination model of Multiple sclerosis in rat* Saeideh Baradarana, Akbar Hajizadeh Moghaddama, Maryam Ghasemi Kasmanb aDepartment

of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran;

bCellular

and Molecular

Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran

Multiple sclerosis (MS) is one of the most autoimmune neurological and inflammatory disease in worldwide. Demyelination and disturbance of action potential conductance are regarded as main signs of MS disease. Hesperetin (Hst) is one of the flavonoid that have neuroprotective properties. The present study attempts to evaluate the effects of hesperetin or its nanoparticle on myelin repair and glial activation in lysolecithin (LPC)-induced demyelination model. Materials and Methods: Local demyelination was induced by administration of LPC (1%, 2μL) into the rat' optic chiasm. Animals have received oral administration of Hst or nano-Hst at dose of 20 mg/kg for 14 and 21 days post lesion. Visual evoked potential (VEPs) records were performed on days 0, 7, 14 and 21 post lesions. Immunostaining against Iba 1 (microglia marker) and GFAP( astrocytes marker) were carried out for evaluation of myelination and astrocytes activation. Results: Electrophysiological evidence emphasize that oral administration of hesperetin and nano-hespretin could reduce the P1N1 latency and increase the amplitude of VEPs waves compared to the saline and Hst groups (p≤0.001). Immunostaining showed that myelin repair was improved in animals which have received nano-Hst treatment; In addition, nano-hesperetin and its nanoparticle effectively reduced the expression of GFAP in optic chiasm(p≤0.001). The extent of demyelination was reduced in animals under treatment of hesperetin p≤0.01, nanohesperetin(p≤0.001). Conclusion: Our results showed hesperetin and nano-hesperetin treatment significantly enhances myelin restoration through endogenous sources of glial progenitor cells following local injection of LPC. Keywords: Myelin repair _ Lysolecithin _Optic chiasm_ hesperetin NPs _ gene expression

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Ablating choroid plexus epithelial cells in the adult mouse brain: A new tool to approach CSF disorders* Alicia M. Bedolla Neuroscience, Molecular Genetics, University of Cincinnati

Cerebrospinal fluid (CSF) has multiple facets for maintaining a healthy brain, such as distributing substances across the parenchyma and clearing waste. Abnormal CSF volume is associated with developmental brain disorders, like pediatric hydrocephalus and autism. Choroid plexus (CP) atrophy has also been described in adults with executive dysfunction. Contrary to its essential roles, the CP-CSF system has remained an under-explored field in neuroscience due to limitations in the number of available tools to precisely modulate development and function. Therefore, development and characterization of robust models that allow manipulation of CSF will have a significant impact on understanding the role of CSF in health and diseases. While exploring microglia ablation models, we unexpectedly discovered a robust tool that can break new ground for studies of the CP-CSF system in the mouse brain. Our preliminary data demonstrate that the transgenic animal with a floxed-diphtheria toxin receptor in the ROSA26 allele (DTR) express sufficient level of DTR exclusively in the CP epithelial cells in the absence of Cre, which induces significant loss of CSF and shrinkage of ventricular space upon diphtheria toxin (Dtx) administration, which was validated by 3D-fluid sensitive magnetic resonance imaging and histological analysis. Furthermore, TUNEL staining demonstrated specific CP epithelial cell apoptosis upon Dtx treatment in DTR mice but not wt controls. Using immunohistochemistry and a battery of behavioral assays, our data suggests disrupted CSF production and circulation leads to progressive neuroinflammation in the adult mouse brain supporting the important role of CSF in maintaining the homeostasis of the brain.

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Extracellular matrix protein network in valvular heart disease Rihab Bouchareb New York, NY

Calcified aortic valve disease affects over six million Americans and is associated with changes in valve leaflets' mechanical properties, resulting in impaired valvular blood flow. CAVD is a fibrocalcific disease with multifactorial risk factors related to the complex environment where the aortic valve (AV) resides. Clinical studies showed that obesity accelerates the progression of aortic stenosis. However, lipid-lowering drugs (statins) didn’t show any efficacy in slowing the development of the disease. Currently, there is no viable pharmacological treatment to stop the disease's progression or activate mineral regression. Extracellular matrix proteins (ECM) are the main component of aortic valve tissue. ECM proteins play an important role in maintaining the physiology and the normal function of the valve tissue. We have examined the ECM proteins network by mass spectrometry and RNA sequencing on 33 human explanted aortic valves. Our network analysis demonstrated a complex linkage between ECM, metabolic complement, and lipid transporter proteins. We identified FNDC1 and MXRA5 as novel ECM markers in calcified valves, selecting them as possible pharmacological targets to slow the progression of aortic stenosis.

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Characterization of the Anti-Müllerian Hormone Prodomain Dimerization State Alena S. Bruening, Kaitlin N. Hart, James A. Howard, David Pépin, Patricia K. Donahoe, Thomas B. Thompson University of Cincinnati, Cincinnati, OH

Anti-Müllerian Hormone (AMH) is an extracellular signaling protein that helps regulate fetal reproductive differentiation and folliculogenesis. Like other members in the Transforming Growth Factor β family, AMH is synthesized as a precursor with an N-terminal prodomain that is proteolytically separated from the C-terminal mature domain, resulting in a complex consisting of dimeric AMH mature noncovalently associated with dimeric prodomain. While the AMH mature domain has been structurally resolved, little information on the structure of the prodomain exists despite its unique size and apparent importance for signaling. The prodomain monomer has five total cysteines (C55, C103, C188, C241, C411) thought to form multiple disulfide bonds integral to its structure and function. The odd number of cysteines leaves at least one free to form a homodimer, which we hypothesize is mediated by cysteine 55. To test this hypothesis, we are using mutational analysis alongside protein fingerprinting. Our initial mutagenesis experiments, where each cysteine was mutated to serine to disrupt disulfide bonds, suggests that C55 forms an intermolecular disulfide bond and that C103 and C188 are an intramolecular disulfide pair. Interestingly, C411S showed an increase in AMH mature secretion. Given that the intermolecular disulfide bond is likely the most sensitive to reduction we identified conditions to minimally reduce and chemically tag the prodomain’s intermolecular disulfide bond for characterization by protein fingerprinting. This work will help determine the structure of the AMH prodomain, ascertain the number, type, and function of the prodomain’s disulfide bonds, and provide molecular information for rational therapeutic design.

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Incorporation of magnesium and elastin in electrospun chitosan membranes for skin wound applications* Alex Bryan1, Andreu Blanquer2, Lucie Bačáková2, Joel D. Bumgardner1 1Department of Biomedical Engineering, UofM-UTHSC Joint Graduate Program in BME, Memphis, TN, USA; 2Laboratory of Biomaterials and Tissue Engr., Institute of Physiology, Czech Academy of Sciences, Prague 4, CZ

Electrospun, chitosan membranes (ESCM) have seen success for guided bone regeneration applications [1]. Chitosan is a biodegradable, naturally occurring polysaccharide derived from crustacean exoskeleton that has many pro-healing properties applicable to other tissue. This biomaterial can also be mixed with another polymer, like elastin, to improve mechanical properties and bioactivity, increasing its healing capabilities [2]. Specifically, the elastin-polysaccharide nanofiber structure may serve as a template in skin tissue engineering applications. A big challenge facing large skin defect healing is the lack of vascularization into the wound. Magnesium (Mg2+) has anti-inflammatory effects and has been shown to play a role in angiogenesis [3]. The goal of this work is to evaluate incorporation of amorphous Mg2+phosphate nanoparticles (MgNP) and elastin into electrospun chitosan membranes for potential use in skin wound applications. Membranes were characterized for nanofiber size and structure, Mg2+ incorporation and in vitro release, elastin incorporation, degradation profiles, and in vitro cytocompatibility. Energy dispersive spectroscopy and in vitro release confirmed successful incorporation of MgNP. Immunofluorescence staining confirmed the incorporation of elastin in ESCMs. Elastin incorporation accelerated membrane degradation in a simulated body fluid containing 300 µg/mL lysozyme. Elastin incorporated membranes also showed improved cytocompatibility when cultured with NIH3T3 fibroblasts over 5 days. This data confirms the incorporation of magnesium and elastin into ESCMs and suggests this novel material may have potential for use in skin wound applications. Current ongoing works include modifying magnesium incorporation and eventual testing to assess angiogenic potential.

[1]

V. P. Murali et al., doi: 10.1111/JRE.12883

[2]

H. Su et al. doi: 10.3390/MD19030169

[3]

D. Bernardini et al. doi: 10.2741/1610

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Cingulate cortex neuroadaptations in a female rat model of combined Complex Regional Pain Syndrome and alcoholic neuropathy* Jessica A. Cucinello-Ragland, Roshaun Mitchell-Cleveland, W. Bradley Trimble, Kimberly N. Edwards, Patricia E. Molina, Liz Simon Peter, Scott Edwards Department of Physiology, LSU Health Sciences Center, New Orleans, LA

Complex Regional Pain Syndrome (CRPS) is an amplified musculoskeletal pain condition that develops following limb injury or immobilization and can persist long after the injury has healed or immobilization has ceased. Although the exact mechanisms underlying the long-lasting nature of the syndrome are unknown, CRPS is associated with central nervous system dysregulation and peripheral hyperalgesia. These symptoms are also observed in alcoholic neuropathy, leading us to hypothesize that these conditions may be pathophysiologically accretive. Additionally, people assigned female at birth appear to be more sensitive to both CRPS and alcoholic neuropathy. To investigate the biobehavioral mechanisms underlying these two conditions, we utilized a model of combined CRPS and alcoholic neuropathy in adult female rats (n=4-5/group). In order to determine the effect of circulating ovarian hormones on these pathologies, half of the animals received an ovariectomy (OVX) at the start of the experiment. To model alcoholic neuropathy, animals were fed either a Leiber-DeCarli alcohol liquid diet or a control diet for 10 weeks. CRPS was modeled in all animals via unilateral hind limb immobilization for 7 days, and mechanical hyperalgesia was measured using the von Frey test 3 days following cast removal. We found that cast immobilization and chronic alcohol both separately and additively decreased mechanical nociceptive thresholds, indicative of hyperalgesia. Interestingly, OVX had no effect on mechanical sensitivity. We next used Western blot analysis to investigate phosphoprotein levels in the cingulate cortex, a limbic region that mediates the affective components of pain. Chronic alcohol increased phosphorylation of glutamatergic receptor subunits GluR1 and NR1 as well as extracellular signal-regulated kinase (ERK) and cAMP-response element binding protein (CREB). Again, we did not observe any OVX effects on protein phosphorylation. Collectively, these findings suggest that chronic alcohol in the context of CRPS facilitates hyperalgesia via hyperexcitability in the cingulate cortex, establishing a novel therapeutic strategy for these conditions.

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Regenerative medicine and Tissue engineering: importance of cells, biomaterials, in vitro and in vivo models Madhu Dhara, Tena Ursinia, Darryl Millisb, Henry S. Adaira, and David E. Andersonc aDepartment

of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville USA;

bDepartment

of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, USA;

cOffice

of Research and Graduate Studies, College of Veterinary Medicine, University of Tennessee, Knoxville USA

Success of regenerative medicine and tissue engineering strategies is dependent on a program, which involves the isolation and characterization of viable cells; design and fabrication of optimal biomaterials and scaffolds; establishment and tools to study appropriate in vitro and in vivo animal models. The tissue regeneration program at the University of Tennessee located in Knoxville, TN brings together an interdisciplinary group of scientists (cell biologists, equine and canine clinicians, material scientists) focused on using adult mesenchymal stem cells for the treatment of diseases. These include vascular, neural, and orthopedic diseases in large (horses) and small (dogs) animals. The long-term goal is to make basic discoveries and to translate these discoveries into the development of diagnostic modalities and treatment protocols to solve complex medical problems related to musculoskeletal, and nerve injuries. We carry out specific in vitro assays to evaluate cell adherence, proliferation, and cellular mechanisms of differentiation into osteocytes, chondrocytes, or neural-like progenitors in a tissue culture setting. We then conduct controlled studies, using rodents, to confirm the biocompatibility and efficacy of mesenchymal cells used alone or in combination with biomaterials. Next, we translate these findings into controlled, preclinical studies using goats, sheep, pigs, horses, and dogs. Ultimately, studies are translated into efficacious treatment options for human patients.

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The interaction between Human Origin Recognition Complex Subunit 1 (413- 511) and G-quadruplex Forming DNA Afaf Eladl1,2,3*, Yudai Yamaoki1,2, Shoko Hoshina4, Haruka Horinouchi4, Keiko Kondo1, Shou Waga4, Takashi Nagata1,2 Masato Katahira1,2 1Institute of Advanced Energy, Kyoto University; 2Graduate School of Energy Science, Kyoto University; 3Faculty of Pharmacy, Zagazig University; 4Department of Chemical and Biological Sciences, Japan Women's University

Hexameric origin recognition complex (ORC) binds to replication origins and has a role in initiation of DNA replication. It is well known that yeast ORC recognizes replication origins in sequence specific manner, while human ORC (hORC) recognizes replication origins sequence non-specifically. In the previous study, we revealed that hORC binds to G-rich DNAs that form G4 structures (G4 DNAs) and a region comprising amino acid residues 413–511 of hORC subunit 1 (hORC1413–511) is important for the binding. Here, we investigated the binding-mode of hORC1413-511 to G4 DNAs. The titration experiments were performed by fluorescence anisotropy measurements, by which the dissociation constants (Kd) of the binding between the hORC1413-511 and DNAs were determined. These results showed that hORC1413-511 preferably binds to G4 structures over double-stranded DNA structures. Further investigations by NMR and CD measurements revealed that those G4 structures retain the G4 structures even after binding with hORC1413-511. Moreover, NMR analysis indicated that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511 and the electrostatic interaction might be the driving force of the interaction between the hORC1413-511 and G4 DNAs. Our results suggest that hORC1 may recognize replication origins through the G4 structure to initiate DNA replication.

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Chronic Inhaled Nicotine-Induced Pulmonary Hypertension and Right Ventricular Remodeling are Mediated by Angiotensin-II Type 1 Receptor* Nicholas D. Fried1, Tamara M. Morris1,2, Anna Whitehead1, Eric Lazartigues1,2, Xinping Yue1, Jason D. Gardner1 1LSUHSC – New Orleans; 2Southeast Louisiana Veterans Health Care Systems

Introduction: Use of electronic cigarettes is rapidly increasing among youth and young adults, but little is known regarding the long-term cardiopulmonary impacts of these nicotine-containing devices. Our group previously demonstrated that chronic inhaled nicotine induces pulmonary hypertension and right ventricular (RV) remodeling in mice, characterized by increased RV systolic pressure (RVSP) and RV free wall thickness during diastole (RVFWT;d). These changes were associated with upregulated RV angiotensin converting enzyme (ACE), leading us to hypothesize that these nicotineinduced effects are mediated by the renin-angiotensin system. ACE inhibitor and angiotensin-II receptor blocker use in a large retrospective pulmonary hypertension patient cohort was associated with improved survival. Here, we utilized losartan, a specific antagonist of the angiotensin-II type 1 receptor (AT1R), to further explore nicotine’s cardiopulmonary effects. Methods: Male C57BL/6 mice received either nicotine vapor or room air for 12 hours per day, and exposure was measured by serum cotinine (559±57 ng/mL; comparable to human cigarette smokers or electronic cigarette users). A subset of mice was treated with losartan via osmotic mini-pump for 8 weeks. Losartan dosage was approximately 5.0 to 6.5 mg/ kg/day due to body weight differences throughout the experiment. Cardiac structure and function were assessed using echocardiography and RV catheterization. Results: Chronic inhaled nicotine significantly increased RVSP (40.7±3.5 mmHg, n=11) versus air exposure (25.6±1.0 mmHg, n=11, p<0.005). Mice receiving both losartan and nicotine had significantly reduced RVSP (24.0±1.3 mmHg, n=12, p<0.005) versus nicotine alone. There was no difference in RVSP between air mice and either losartan-infused group (air-losartan: 23.7±1.2 mmHg, n=14). RVFWT;d was increased in nicotine mice (0.44±0.01 mm, n=12) versus air mice (0.31±0.01 mm, n=10, p<0.0001). Losartan infusion of nicotine mice resulted in significantly decreased RVFWT;d (0.34±0.01 mm, n=8, p<0.0001) versus nicotine alone. There was no difference in RVFWT;d between air mice and either losartan-infused group (airlosartan: 0.32±0.01 mm, n=7). Chronic inhaled nicotine increased RV internal diameter during diastole (RVID;d; 1.56±0.04 mm, n=12) versus air mice (1.24±0.07 mm, n=10, p<0.0001), suggesting RV dilation. RVID;d between air- exposed mice receiving losartan (1.23±0.04, n=7) and nicotine-exposed mice receiving losartan (1.44±0.03, n=8) was not significantly different. Neither nicotine nor losartan exposure resulted in changes to left ventricular structure and function. Conclusion: These findings provide the first preclinical evidence that AT1R antagonism can ameliorate chronic inhaled nicotine-induced pulmonary hypertension and RV remodeling.

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Efficiency of Odontoblasts Generated from Induced Pluripotent Stem Cells and Gingival Mesenchymal Stem Cells Kirollos Gerges, Layla Norbash, Patrick B. Nabholz, Sheeja Rajasingh, Narasimman Gurusamy, Vinoth Sigamani, Daniel Brown, Mustafa Dabbous and Johnson Rajasingh Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, TN

Introduction: Mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into different kinds of cells that can be used to treat many illnesses. Odontoblasts are the major cell source for tissue engineering and experimental models to investigate tooth growth and differentiation. One of the diseases that odontoblasts can potentially treat is dental caries, and we believe this can be done through regenerating odontoblasts, which is responsible for the formation of dentin. We have generated odontoblast through two different sources. Our aim is to compare the odontoblasts (G-ODO) differentiated from gingival MSCs (G-MSCs) and odontoblasts (G-iODO) differentiated from induced pluripotent cells derived from G-iMSCs. Hypothesis: The odontoblasts derived from gingival induced pluripotent stem cells (G-iODO) are the better source of cells than those that are derived directly from G-MSCs. Methods and Results: The levels and presence of odontoblast specific genes and proteins were compared across two different odontoblasts by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and immunostaining analyses. Our results showed that alkaline phosphatase (ALP), secreted phosphoprotein 1 (SPP1) and dentin matrix acidic phosphoprotein 1 (DMP1) were highly expressed in the G-iODO than G-ODO. We also confirmed the lack of the expression of pluripotency markers such as OCT4 and Nanog in G-iODO. Conclusion: We do report that G-iODO is a better and more viable source of cells than G-ODO for dental regenerative therapy.

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Exploring the unique biochemistry of an artificial BMP heterodimer Gregory R. Gipson, Kristof Nolan, Chandramohan Kattamuri, Thomas B. Thompson University of Cincinnati, Cincinnati, OH

Historically most research into the signaling proteins of the TGF-beta family has been focused on homodimeric proteins. However, recent studies performed on heterodimeric BMP2/BMP7, BMP2/BMP6 and BMP9/BMP10 have illustrated the importance of these heterodimeric proteins within the context of TGF-beta signaling. To expand the study of BMP heterodimers, we have determined that mature GDF5 can be combined with mature BMP2 to form a BMP2/GDF5 heterodimer. Intriguingly, this combination of a BMP2 monomer with high affinity to heparin sulfate with a GDF5 monomer with low heparin binding affinity produces a heterodimer with an intermediate heparin binding affinity, less likely to be bound to the extracellular matrix and thus with greater potential for endocrine signaling than is typical for BMPs. This procedure was then repeated with BMP4, yielding a BMP4/GDF5 heterodimer. These heterodimers were then characterized using both biochemical and biological approaches to demonstrate that they signal more robustly than their respective homodimer parents, likely driven by an increased affinity for their type 1 receptors. Furthermore, the crystal structure of BMP2/GDF5 heterodimer was solved, showing modified type 1 receptor binding sites

P21

The experimental validation of gene-gene fusions as a diagnostic tool for the detection of Glioblastoma Multiforme (GBM)* Olawumi D. Giwa, Gidi Baum, Milana Frenkel-Morgenstern Cancer Genomics and BioComputing of Complex Diseases Lab, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel

Gliomas are tumors with etiologies in glial cells resident in the brain. They account for approximately 80% of all malignant brain tumors. High-grade gliomas such as glioblastoma multiforme (GBM) are the most co mmon types of primary malignant brain tumors. The standard treatment paradigm for patients with GBM is still very limited in terms of survival. Therefore, we selected GBM as a model to investigate and subsequently develop an early non- invasive (liquid biopsies) diagnostic tool and precision medicine methodology to monitor patients using blood plasma-derived circulating cell-free DNA (cfDNA). Our laboratory analyzed cfDNA samples obtained from GBM patients (n=27) and compared them with samples from healthy individuals (n=20). We have demonstrated that the concentration of cfDNA in the plasma of GBM patients is significantly higher than in the plasma of healthy individuals. In addition, Next-generation sequencing (NGS) analysis of the cfDNA revealed several putative chimeric DNA in the GBM samples that are not present in samples from healthy individuals. We are currently trying to confirm and validate the NGS findings by several different methods 1) Probe-based Polymerase Chain Reaction (qPCR) assays on the cfDNA samples in which the probe is located directly on the fusion site; 2) cloning and Sanger sequencing of PCR product/s from the cfDNA; and 3) Oxford NanoPore sequencing. The future aspect of these findings can be used for novel drug targets and precision medicine for GBM patients to improve the patient’s prognostic outcomes.

Keywords: Glioblastoma, liquid biopsy, cell-free DNA, Next-generation sequencing, drug design and development.

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Autologous Induced-Mesenchymal Stem Cells Demonstrate Superior Cardioprotective Characteristics Against Injuries Narasimman Gurusamy, Sheeja Rajasingh, Vinoth Sigamani, Selene G. Perales, Johnson Rajasingh Department of Bioscience Research, University of Tennessee Health Science Center, Memphis, TN

Background: Treatment with adult mesenchymal stem cells (MSCs) is promising for cardiovascular diseases because of their plasticity, replicative potential, immunomodulation, and secretion of paracrine factors. Recently, we have generated autologous induced pluripotent stem cell derived MSCs (iMSCs) from non-invasively obtained human urinary epithelial cells (UE), through employing a safe non-viral method of reprogramming. Hypothesis: We hypothesize that UE-derived autologous iMSCs and their exosomes possess superior cardioprotective and regenerative characteristics when compared to the commonly used adult umbilical cord MSCs (referred as MSCs) for the treatment of ischemic cardiomyopathy (ICM). Methods and Results: We compared the cardioprotective properties of the iMSCs with MSCs for ICM. Our results showed that co-culturing of iMSCs with the human skin fibroblast-derived induced cardiomyocytes (iCMCs) or treatment of iCMCs with exosomes obtained from the conditioned media of iMSCs offered a superior protection of iCMCs from angiotensin-II (Ang, 10 µM for 24 hrs) and hypoxia-reoxygenation (6 hrs of 1% hypoxia followed by 24 hrs of reoxygenation)–mediated injuries, compared with their respective MSC controls. Furthermore, the cardioprotective effects were studied through qRT-PCR and Western blot analyses for the expression of survival genes/proteins such as BCL2, NRF2, and measurement of mitochondrial membrane potentials, intracellular reactive oxygen species, and in situ cell death by apoptosis. Conclusion: Our results indicate that non-invasively obtained autologous iMSCs and their exosomes possess a superior cardioprotective characteristic than adult umbilical cord MSCs and pose as a promising source of cells for the treatment of ICM.

P23

In vivo evaluation of macrophage polarization in response to raspberry ketone-loaded chitosan membranes Melika Esmaeili Rad1, Samantha Hall1, Fernanda Guerra1, K. Mark Anderson2, Omar Skalli3, Jessica A. Jennings1, Joel D. Bumgardner1 1Joint Graduate Program in Biomedical Engineering, University of Memphis-UTHSC-Memphis, Memphis, TN; 2Department of Diagnostic Sciences, College of Dentistry, UTHSC-Memphis, Memphis, TN; 3Department of Biological Sciences, University of Memphis, Memphis, TN

Nanofibrous electrospun membranes made from chitosan have shown promise for enhanced guided bone regeneration in alveolar defects when insufficient bone volume is present. The nanofibrous structure provides an increased surface area to volume ratio that allows for local drug delivery to stimulate healing. A strategy that can be implemented to facilitate healing is the promotion of macrophage polarization from a pro-inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2). Raspberry ketone (RK) is a natural phenolic compound that possesses antioxidant and anti-inflammatory properties. Previous studies have shown that RK has potential to facilitate macrophage polarization. This study used electrospun chitosan membranes (ESCMs) to locally deliver RK to an in-vivo bone defect site using a rat calvarial model. ESCMs were loaded with 0 (control), 100, or 500 µg RK. Membranes from each treatment group were implanted into rat calvarial defects (n=8). Each animal received one control and one ESCM loaded with RK. Membranes and surrounding tissues were extracted in serial sections and immunohistochemically stained at 1, 2, and 4 weeks using individual markers for M1(iNOS), M2 (CD206), and total macrophages (CD68). Images of the stained tissues were obtained, and the percentstained area was quantified using NIH ImageJ. Analysis was performed by a blinded observer. Results indicated that ESCMs loaded with 100 µg RK facilitated the M1 to M2 polarization in comparison to the 500 µg RK or control groups. Therefore, RK shows promise for the promotion of bone healing.

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In silico modeling of the AMH prodomain reveals unique features within the TGF-β family* James A Howard Pharmacology and Systems Physiology, University of Cincinnati

Anti-Müllerian hormone (AMH) is a signaling ligand within the transforming growth factor β (TGF-ß) family important for development of the reproductive organs and folliculogenesis in females. Like all TGF-β family members, the mature domain of AMH is synthesized as a single polypeptide containing a large N-terminal prodomain, then proteolytically cleaved to form a noncovalent complex necessary for biological activity. For some ligands of the family, the prodomain noncovalent complex renders the signaling molecule latent while in others the function is unknown but is thought to be protective. For AMH, the prodomain has been shown to be indispensable for biological function. Structures of prodomain-ligand complexes show a variety of binding modes and interactions. Interestingly, the AMH prodomain is the largest prodomain within the family and is predicted to have unique features, however the structure remains unresolved. In the absence of an experimentally derived structure and to better understand the interaction of the prodomain with mature AMH, we have used AlphaFold to generate several models of the AMH prodomain, which are supported by preliminary small angle x-ray scattering (SAXS), negative stain electron microscopy data, and mutagenesis. From these observations, we predict that the AMH prodomain has a unique dimerization cysteine that links two prodomain chains. Further, the prodomain seems to be divided into an N-terminal and a C-terminal lobe connected by a proline-rich linker region, much different than other prodomains of the family. Future experimental effort will be directed towards validating these models and resolving the structure of the AMH prodomain complex.

P25

Senescence-Associated Secretory Phenotype (SASP) factors down-regulate glutamate transporter expression in human brainstem astrocytes through a paracrine fashion* Manoj Jagadeesh1, Mahesh Kumar Sivasubramanian1, Raisa Monteiro1, Priya Balasubramanian2, Madhan Subramanian1 1Oklahoma State University, College of Veterinary Medicine Stillwater, OK, United States; 2University of Oklahoma Health Sciences Center

Glia-neuron crosstalk in the brainstem is emerging as a critical player in the regulation of sympathetic nerve activity (SNA). Specifically, astrocytes modulate SNA by tightly regulating synaptic glutamate levels through transporter mediated re-uptake mechanisms. Pro-inflammatory mediators like TNFα have been shown to negatively affect glutamate transporter expression in astrocytes. Previous studies from our laboratory have provided evidence for oxidative stress-induced senescence and their secretory phenotype (SASP), a potential source of inflammation, in human brainstem astrocytes. Hence, in the present study we hypothesize that inflammatory mediators in SASP factors down-regulate glutamate re-uptake transporter expression in a paracrine manner. To test our hypothesis, we induced senescence in cultured human brainstem astrocytes with hydrogen peroxide (H2O2) treatment at a dose of 300 μM for 2 hours. Senescence induction was confirmed through PCR expression for senescence markers and SA-beta-gal staining 7 days after H2O2 treatment. Conditioned media collected from the senescent and control astrocytes were used to assess the paracrine effect of SASP factors on astrocytic glutamate transporter expression. Following 36 hours of treatment with conditioned media from senescent astrocytes, we observed a significant down-regulation of two of the major astrocytic glutamate re-uptake transporters, EAAT1 (GLAST-1) and EAAT2 (GLT-1) in human brainstem astrocytes. This suggests that soluble SASP factors in the secretome of senescent cells can alter the function of glutamate transporters in the astrocytes through a paracrine action. Increased activity of glutamatergic neurons in the rostral ventrolateral medulla due to reduced glutamate re- uptake at the synapse may be a possible glial mechanism underlying enhanced sympathoexcitation in obesity.

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P26

Molecular Characterization of the Inhibin Heterodimer and its role in Activin Antagonism* Kappes E.C., Kattamuri C., Czepnik M., Thompson T.B. Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH

Inhibin, a Transforming Growth Factor-Beta (TGF-Beta) antagonist, is an endocrine factor that is responsible for suppressing the follicular stimulating hormone at specific intervals. Inhibin is critical for maintaining folliculogenesis which promotes reproductive health in both men and women. Unlike the other TGF-Beta ligands, inhibin is a heterodimer made up of two subunits, alpha and beta. This unique protein structure allows inhibin to block and thereby regulate activin homodimer (beta) signaling without producing a signal of its own. The working model for activin antagonism is that the inhibin beta subunit binds and sequesters one activin receptor, reducing receptor availability on the cell surface. This model does not describe the role of the inhibin alpha subunit nor how this heterodimer formation specifically targets activin signaling complexes. We hypothesize that both inhibin subunits bind and sequester one type of activin receptor each to disrupt the formation of an active signaling complex. The long-term goal is to understand the molecular mechanisms of inhibin heterodimer formation as a model for disease-related inhibition of cellular signaling.

P27

SIN3 complexes regulating the Epigenetic basis of Cell fate specification and Differentiation during Development and Disease Ram Parikshan Kumar1,2, Xinwei Cao2, Soumen Paul1,3 1Department of Pathology and Laboratory of Medicine, University of Kansas Medical Center, Kansas City, KS, USA; 2Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA; 3Institute for Reproductive Health and Perinatal Research, Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, KS, USA

SIN3-histone deacetylases (HDACs)-containing complexes participate to the oncogenic transformation in numerous human cancers. Mammals possess two highly similar proteins SIN3A and SIN3B. Although, these SIN3A and SIN3B are co-expressed in many tissues and function redundantly, these two proteins have also evolved to perform specialized functions. mSin3A deletion in the developing mouse embryo results in lethality at the post-implantation stage with no embryos by e6.5 demonstrating that it is an essential gene. In contrast to mSin3A, mSin3B show perinatal lethality and none of the pups survive at birth. Recently, it has been shown that SIN3A or SIN3B heterozygous patients develop syndromic intellectual disability/autism spectrum disorder. SIN3 knockdown in neuroblastoma cells and breast cancer cells prevents neuronal differentiation and breast cancer metastasis respectively. As SIN3 targets histone deacetylases to chromatin to repress transcription through interacting with sequence specific DNA binding transaction factors and SIN3B is dysregulated in in many human cancer types, the role of SIN3 has not been addressed either in the development of normal BRAIN or PLACENTA or phenotype associated with tumour, impairing the development of these two tissues. My hypothesis is that SIN3 and interacting proteins are required for the progenitor cell fate specification and differentiation. Specifically, how SIN3 and its interactors regulate the epigenetic and epitranscriptomic programming which goes awry in tumors. I want to test this hypothesis using multi-organ system and several types of primary cells derived from patient samples. To explore the function of SIN3B during the nervous system development, I generated Sin3B conditional knockout (Sin3BF/F; Nestin-cre, Sin3BcKO) mouse. Preliminary data suggests that during nervous system development, SIN3B has acquired specific biological function in neural progenitor cells (NPCs) as Sin3BcKO mouse show perinatal lethality most probably due to failure in generation of mature neurons for the initiation and maintenance of respiratory rhythms. Surviving Sin3BcKO animals (6%) are fertile. Specifically, pups born from the Sin3BcKO did not survive after day1 or day2 due to lack the maternal/feeding behavior. This opens new directions to explore the role of SIN3B regulating the neuronal circuit associated with social attachment. Like NPC, shRNA mediated knockdown of SIN3A and SIN3B in human trophoblast stem cells (hTSC) results in impaired stem cell fate and may be concomitant differentiation into syncytiotrophoblast (ST) and extravillus trophoblast (EVT). Depending on the progress on going projects, I will be expanding the research directions with Sin3A, Sin3B and interacting partners associated with RNA metabolism identified using SIN3B RIME. I believe that exploring the molecular mechanism associated with SIN3B using state-of-the-art genomic approaches during development and differentiation of Neural Progenitor cells/Placental Progenitor cells or Stem cells and finding new strategies will be of therapeutic importance.

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P28

An optimized cell-loading approach and its optimization of cellulosic bioinks to improve cell viability in cell-free 3D bioprinting* Zheng Li1, Zhongqiang Li1, Alexandra Ramos2, Shaomian Yao2, and Jian Xu1 1Division of Electrical and Computer Engineering, College of Engineering, Louisiana State University, Baton Rouge, LA 70803, USA; 2Department of Comparative Biomedical Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA

Cell-laden printing is the most commonly used approach in 3D bioprinting, but the major drawback is that cell viability is highly affected by the extrusion pressure in the printing process. In this study, we develop a self-absorbent (SA) deposition to load cells post-cell-free 3D-printing, and optimize bioink by adding extracellular matrix (ECM) and collagens. We prepare bioinks with cellulosic nanofiber and alginate by the ratio of 20:10 (NFA20/10) and 20:02 (NFA20/02). NFA bioink was optimized by adding ECM, and collagen type I and III (COL-I/III), and type II (COL-II): NFA20/02-ECM, NFA20/02COL-I/III, and NFA20/02-COL-II. Next, hFOB cells were loaded to the scaffolds with self-absorbent (SA) cell deposition developed in our lab. Cell proliferation was assessed by alamarBlue assay. Both NFA bioinks had excellent printability and shape fidelity; SA could achieve homogenous cell distribution in 3D-printed scaffolds. NFA 20/02 had better cell viability than NFA20/10. Cell survival and proliferation were greatly improved by adding ECM, and collagens, but extraction of ECM from animal tissues is time-consuming and labor-intensive. COL-I/III and COL-II were the optimal option for the mass experiments; many cells could still reside after 30-days of culture; dual-porous (DP) scaffolds supported cell proliferation better than Inherent-porous (IP) scaffolds. Overall, SA method could achieve a similar cell-loading efficiency to cell-laden printing. NFA20/02-COL-II with SA method show the best cell viability and may serve as an optimal option for our cell-free 3D-bioprinting.

P29

Osteogenic Regulation of Human Mesenchymal Stems on Graphene Amber F. MacDonald, Ruby Trotter, Austin Bow, Amanda Murphy, Lisa Amelse, Tom Masi, Shawn E. Bourdo, Madhu Dhar UTCVM

Background: Degenerative bone diseases imposes a global health care burden every year. Treatment strategies include osteogenic differentiation, thereby creating new bone material. We previously identified that mesenchymal stem cells (MSCs) cultured on graphene supports osteoblast development in vitro. However, the osteo-genetic signaling of MSCs in the presence of graphene is largely unknown. Methods: MSCs extracted from human adipose tissue (AD-MSCs) or human bone marrow (BM-MSCs) were cultured on graphene for 7 or 21 days. At each time point, isolated RNA was reverse transcribed to cDNA before loading onto RT2 Profiler PCR Human Osteogenesis Array. Gene expressions of both cell lines were compared either over time or between both cell lines at each time point. Results: AD-MSCs and BM-MSCs demonstrated osteoblast mineralization in the presence of graphene. We then examined expression of 84 genes that were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Over time, both MSC sources showed significant regulation from each genetic category. However, when BM and AD-MSCs were compared to each other, the BM-MSCs demonstrated a robust response at both time points. Conclusion: AD-MSCs and BM-MSCs cultured on graphene showed significant changes in osteogenic gene expressions. These cells were cultured in absence of chemical inducers, suggesting graphene alone commits osteo-genetic signaling of MSCs. We also observed that osteogenesis of BM-MSCs may occur at an earlier time point than AD-MSCs. Overall, MSCs isolated from two independent tissue sources spontaneously expressed osteogenic genes in the presence of graphene.

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P30

Characterization of the Oligomeric States of Erythroferrone, a TGF-β Antagonist Jacob Mast, Thomas Thompson University of Cincinnati

The transforming growth factor β (TGF-β) superfamily of extracellular signaling ligands is inhibited by a number of structurally diverse antagonists. One such inhibitor, erythroferrone (ERFE), is produced by developing erythrocytes and antagonizes BMP-6, a TGF-β ligand that negatively regulates iron import. After a major blood loss event, ERFE is produced and supports the iron update needed for the maturation of erythrocyte precursors. Additionally, constant expression of ERFE in β-Thalassemia patients will cause iron overload. The protein itself consists of a globular C1Q/TNF-like domain and an unstructured domain with a hydroxylated PGP collagen-like repeat. ERFE is believed to use both domains and interchain disulfide bridges to form trimer, hexamer, and 12-mer species. These different oligomeric forms remain uncharacterized in activity, binding stoichiometry, and structure. Recently, we identified a heparin sulfate binding motif (KKXK) immediately upstream of the collagen-like domain and subsequently utilized this property to purify recombinant ERFE via a heparin affinity column. Curiously, while previous publications reported a dramatic loss of activity after purification, heparin affinity chromatography resulted in a pure and potent BMP-6 antagonist. Purified ERFE was examined via SDS- PAGE and cross linking, which revealed a trimer composed of a disulfide linked dimer and monomer, a configuration not previously reported. This trimer was further analyzed using negative stain EM and analytical ultracentrifugation (AUC) to validate the observations and reveal binding stoichiometry. Understanding these different oligomeric states will help reveal ERFE’s mechanism of action which can lead to treatment of patients with iron regulation disorders.

P31

Increasing the Availability and Quality of Human Tissue in Research Janine McCarthy, Kristie Sullivan Physicians Committee for Responsible Medicine, District of Columbia

Advances in 3D and other in vitro tissue model systems have led to important developments in research on human diseases, advancement of novel therapies, and safety testing. In addition, histological and cellular studies of human tissues continue to serve as keystones in understanding disease and health processes. Technologies, like organs-on-chips and 3D bioprinting, rely on human cells and have the potential to have a powerful impact on scientific advancement. As interest grows in using more human tissues, guidelines are needed to encourage consensus among stakeholders concerned with the use of human tissues and human-model systems, to establish and maintain best practices, to promote effective quality control systems, to facilitate education and training, to support journal editors, and to help end users and regulators who need to understand results and draw conclusions based on human tissue data. A lack of a standardized approach to the donation, procurement, and processing of tissue, coupled with the absence of a U.S. wide strategy to oversee recovery and use leaves gaps in tissue quality and availability. Our working group, comprised of stakeholders from the research community, regulatory agencies, and organ procurement organizations, are exploring and addressing the barriers to the use of human tissues and cells in research. We have established a set of recommendations and are developing guidelines for standardizing the characterization of human tissues and cells to facilitate greater access to high-quality human tissues for biomedical research and toxicology and help ensure the transition away from the dependence on animal models.

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P32

Renin-Angiotensin System Alterations in the Human Alzheimer's Disease Brain Golnoush Mirzahosseini, Saifudeen Ismael, Heba A. Ahmed, Arum Yoo, Modar Kassan, Kafait U. Malik University of Tennessee Health Science Center, Memphis, TN

Alzheimer's disease (AD) is the sixth leading cause of death in the United States. Understanding the pathophysiologicalpathways of AD is essential to unravel the complex nature of the disease and identify potential therapeutic targets. The renin-angiotensin system (RAS) has been implicated in several brain diseases, such as traumatic brain injury (TBI), ischemic stroke, and AD. This study was designed to determine the protein expression levels of the RAS components including angiotensin (Ang) II, angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), angiotensin-converting enzyme 1 (ACE-1), and angiotensin-converting enzyme 2 (ACE-2) in postmortem cortical and hippocampal samples of human brains from individuals who had AD or non-AD, using immunoblotting and immunohistochemistry. Our results show that the expression of AT1R increased in the hippocampus, whereas AT2R expression remained almost unchanged in the cortical and hippocampal regions of AD brains compared to non-AD brains. However, Mas receptor is downregulated in the hippocampus. We also detected slight reductions in ACE-1 protein levels in both the cortex and hippocampus of AD brains, with minor elevations in ACE-2 in the cortex. We did not find remarkable differences in the protein levels of Angiotensinogen (ANG) and Ang ΙΙ in either the cortex or hippocampus of AD brains, whereas we observed a considerable increase in the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. The current findings support the significant contribution of RAS components in AD pathogenesis, further suggesting that strategies focusing on the AT1R and AT2R pathways may lead to novel therapies

P33

Comparison of Anti-inflammatory Properties of Induced Mesenchymal Stem Cell-Derived from Periodontal Ligament Fibroblasts and Umbilical Cord Mesenchymal Stem Cells* Patrick B. Nabholz Dentistry, University of Tennesseee Health Science Center College of Dentistry, Memphis, TN

Introduction: Mesenchymal stem cells (MSC) are multipotent cells and have a role in tissue repair, particularly via their anti-inflammatory properties. For years, storing of umbilical cord MSCs (UC-MSCs) has been used across the world as a source of cells for future therapy. This method, however, is expensive and the shelf-life of stored UC-MSCs remains unknown. However, the patient-specific somatic cells are reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into MSCs referred as induced MSCs (iMSCs), offers a better source of cells for therapy. Hypothesis: We hypothesize that the iMSCs derived from human periodontal ligament fibroblasts (hPDLF-iMSCs) is an effective and alternative source of cells for therapy when compared to the UC- MSCs in relation to the anti-inflammatory properties. Methods and Results: hPDLF-iPSCs, available at Dr. Johnson’s lab, were used for generating iMSCs. The UC-MSCs were purchased and maintained under specific culture conditions. Our qRT-PCR data showed that both hPDLF-iMSCs and UCMSCs expressed high levels of mRNA of MSC specific markers CD105 and CD 73 and low levels of pluripotency markers Oct4 and Nanog. Western blot and immunofluorescent analysis revealed high levels of protein expression of CD105 and CD73 in both hPDLF-iMSCs and UC-MSCs. qRT-PCR expression of anti-inflammatory markers such as IL11, TGFB and TSG6 showed that the anti- inflammatory properties of hPDLF-iMSCs were similar to those of UC-MSCs. Conclusions: hPDLF-iMSCs appear to have anti-inflammatory properties similar to those of UC-MSCs and provide an alternative or better source of cells for cell therapy.

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P34

Study of 3D printed poly(lactic co glycolic acid) carbonbased nanomaterial to repair a femoral critical sized segmental defect utilizing Micro-CT evaluation Steven D. Newby1, Austin Bow1, Shawn E. Bourdo2, Andrew Gross3, Joseph Cheever3, Ryan Moffat3, Man Hung3, Madhu S. Dhar1 1Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA; 2Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, USA; 3Roseman University of Health Sciences, College of Dental Medicine, Salt Lake City, UT, USA

Defects within the long bones of patients from serious complications can be extensive in damage. The design and implantation of a biofabricated biocompatible 3D layer by layer construct into the nonunion, which can promote bone and vasculature, is imperative. Microcomputed tomography (micro-CT) is a fundamental mechanism for evaluating biomaterialosteo remodeling and regeneration, it is possible to access data about the tissue’s 3D microarchitecture and distribution in the region of interest. In this study MicroCT evaluation, digital radiography and histological chemistry analysis was used to demonstrate bone regeneration induced by two scaffolds iterations of poly(lactic-co-glycolide) (PLGA) [Mix 1 50:50+65:35] and [Mix 2 - 50:50+75:25] each blended with carbon-based nanomaterials using acellular (n=12) and cellular (n=12) with human adipose derived stem cells (hADSC) in a 5mm segmental femur defect rat model. Micro-CT analysis was completed for visualization of entire rat femur prior to histological examination to improve sectioning orientation and to compare bone volume to tissue volume ratios (BV/TV) at four different thresholds of the Regions of Interest (ROI) after animals were sacrificed and femurs harvested 60 days post-implant. This study demonstrates that PLGA with carbonbased nanomaterials might be useful to promote bone regeneration.

P35

On the Mathematical Models for Cancer Growth Sorush Niknamian Libery University

Cancer disease is the second cause of death in the United States and world-wide. Most Researchers estimate that 595,690 of American people will die from cancer at the end of the year 2017. That means 1,600 deaths/day approximately. Cancer in modern societies is commonly treated with the combination of organ surgery, chemotherapy and radiotherapy. Many kinds of diet strategies have been experimented. However, none of them have been particularly effective. Interestingly, there is some applied research suggesting that a very low-carb ketogenic diet may help. According to Otto Warburg hypothesis, the cause of cancer is the change in the metabolism of mitochondrion in human cells. Low oxygen in tissues in combination with high blood glucose will change the cell respiration from aerobic to anaerobic which leads to fermentation type of respiration. In this research we have collected some useful mathematical models and describing the best model for cancer growth.

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P36

Machine learning approach to integrate multitranscriptomic datasets to identify biomarkers related to Immune thrombocytopenia (ITP)* Haseeb Nisar Institute of Biochemistry and Biotechnology, University of the Punjab

Immune thrombocytopenia (ITP) is an autoimmune disease where the autoantibody opsonized platelets are destroyed prematurely in the reticuloendothelial system and directly by cytotoxic T-cells and complement activation. It is currently impossible to predict the long-term outcome with a high degree of certainty in a case of newly diagnosed ITP. With the advent of Machine learning (ML) as a big data tool, which is not yet fully explored, we can use the different biological datasets to identify pattern recognition in complex datasets derive from genome or transcriptome sequencing. The study uses transcriptomic data derived from either microarrays or RNA-Sequencing and analyze the expression pattern to identify functionally relevant groups of genes under different conditions or phenotypic categories. Datasets obtained from public databases were obtained as raw data to identify differentially expressed genes which will be used later using the systems biology approach to determine the best regions for drugs targeting. We uses supervised and unsupervised Machine learning (ML tools) with a powerful analytical approach to combine several multi-transcriptomic datasets. We concluded that such a novel computational approach could reveal the main gene expression patterns of acute and chronic ITP.

P37

Comparing the Immunomodulatory Effects of Gingival Mesenchymal Stem Cells and Rejuvenated Mesenchymal Stem Cells Layla Norbash, Patrick B. Nabholz, Kirollos Gerges, Sheeja Rajasingh, Narasimman Gurusamy, Vinoth Sigamani, Daniel Brown, Mustafa Dabbous and Johnson Rajasingh University of Tennessee Health Science Center, Memphis, TN

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells, capable of differentiating into multiple types of cells. MSCs also have the potential to provide a great deal of expandability, hypo-immunogenicity and the anti-inflammatory properties that are promising for tissue regeneration. Studies have shown that induced pluripotent stem cells (iPSCs)derived MSCs provide an excellent source of cells for patient-specific autologous cell therapy. Hypothesis: The gingival iPSC derived rejuvenated MSCs (rMSCs) is an excellent source of cells for therapy when compared to the MSCs derived directly from gingival tissues (G-MSC) in relation to the anti-inflammatory properties. Methods and Results: GMSC-iPSCs, available at Johnson’s lab, were used for generating rMSCs and isolated directly from gingival tissues (G-MSCs) were cultured under specific-conditions. Our qRT-PCR and western blot results showed that the mRNA and protein expressions of CD73 and CD105 were highly expressed by the rMSCs compared to the GMSCs. Besides, the mRNA expressions of anti-inflammatory genes such as interleukin 11 (IL11), transforming growth factor-β (TGFβ) and TNFα-stimulated gene-6 (TSG6) were significantly increased in rMSCs compared to the G-MSCs. On the other hand, the expressions of iPSC-specific markers Oct and Nanog were not found in rMSCs. The wound healing property of the MSCs were studied the scratch assay, which showed that both rMSC and G-MSC had comparable migration and healing capabilities. Conclusions: The rejuvenated MSCs derived from G-MSC-iPSCs possess improved anti-inflammatory properties compared to the directly isolated G-MSCs and offer a better and alternative source of autologous cells for treating gingival diseases.

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P38

The A2 Axis: ABI1 and AR reciprocal regulation in prostate cancer* Porter B.A., Hushaw S.H, Kukkonen K., Bah A, Urbanucci A, Zhang F, Fazli L, Gleave M, Bratslavsky G, Kotula L. SUNY Upstate Medical University, Syracuse, NY

Prostate cancer (PCa) is a slowly progressing disease that affects nearly 50% of males over the age of 60. PCa treatments primarily target the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. ABI1 is a putative tumor suppressor and scaffold protein of the WAVE Regulatory Complex (WRC). ABI1 also contains androgen receptor binding sites within its 2nd intron and binds to AR. In this study we aim to characterize the ABI1-dependent AR regulation in PCa. We generated an ABI1 KO in the LNCaP cell line using CRISPR-Cas9. We then preformed transfection assays with fluorescently tagged proteins, ABI1-mcherry and AR-YFP, to visualize the localization using confocal imaging. As a control we compared localization of ABI1-mcherry with ABI1-HA tagged protein that is known to not impede function. We were able to visualize ABI1 localization in cells using fluorescently labeled ABI1 in an antibody independent system. We compared the localization of different ABI1 isoforms and observed slight nuclear changes between isoforms. We then preformed co-transfection of YFP-AR and ABI1-mCherry and observed co-localization. Our studies demonstrate that AR and ABI1 co-localize to the nucleus. Additionally, AB1 isoforms can differentially localize to cytoplasmic and nuclear cellular compartments indicating alternative isoform function with AR. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and AR localization to promote EMT through downregulation of ABI1. These findings will allow for novel insights into the mechanisms underlying AR and ABI1 relationship in neoplastic progression.

P39

Roles of Cysteine-rich Secretory Protein LCCL Domain Containing 2 (CRISPLD2) in Differentiation of Human Mesenchymal Stem Cells Weiqiong Rong, Calvin Rome, Shaomian Yao* Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

Human mesenchymal stem cells (hMSCs) are promising cell sources for tissue engineering and regenerative medicine, primarily owing to their multilineage differentiation potential. Long-term in vitro expansion of hMSCs is essential to obtain sufficient cells for their therapeutic applications; however, such expansion leads to impaired multipotency of hMSCs, which is a major roadblock for their clinical use. This study aimed to investigate the molecular mechanisms causing the impairment of multipotency of hMSCs during expansion. RNA-seq and RT-qPCR analyses identified that 25 downregulated genes were shared by late passage human bone marrow stem cells (hBMSCs), human adipose stem cells (hASCs), and human dental pulp stem cells (hDPSCs). Of them, CRISPLD2 was the most downregulated, with over a 90% decrease in late passage (P11) compared to early passage (P3) cells. Knockdown of CRISPLD2 by transfection of early passage hBMSCs with siRNA significantly abolished the mineralization ability of the cells, as shown by Alizarin Red S staining and calcium quantification, and led to decreased expression of osteogenic marker genes, alkaline phosphatase (ALP), and collagen type I alpha 1 (COL1A1). Additionally, adipogenic differentiation assay revealed that knockdown of CRISPLD2 enhanced adipogenesis of hBMSCs, as determined by Oil Red O staining and RT-qPCR showing increases of adipocytes and expression of adipogenic marker genes, peroxisome proliferator- activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Collectively, CRISPLD2 plays a vital role in maintaining the homeostasis of hMSCs multipotency. Decreased CRISPLD2 expression in hMSCs during in vitro expansion can cause the cells to lose osteogenic differentiation ability.

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P40

SARs-CoV-2 Sequencing: Protocol Optimization to Improve Sequencing Success Rate Samirah Saiid, Baaba Quansah, Fred Tei-Maya, Collins Misita, Dominic Amuzu, Lucas Amenga- Etego, Peter Quashie, Yaw Bediako, Gordon Awandare , Joyce Ngoi West African Centre for Cell Biology of Infectious Pathogens, WACCBIP, Department of Biochemistry, Cell & Molecular Biology, University of Ghana, Legon, Accra, Ghana

The emergence of the novel SARS-CoV2 variants poses great public health threats against efforts being made to eradicate the virus. There has been over 98,000 confirmed cases of the corona virus disease 2019 (COVID-19) in Ghana, and 806 recorded deaths as of 17th July, 2021(WHO), with increased morbidity and mortality as well as transmission been associated with emerging strains of the virus. Genomic surveillance of SARs-CoV-2 plays an important role in understanding the epidemiology and transmission of the virus, bringing together sequencing technologies to the forefront and the need for robust protocols. Here, we present an optimized RNA extraction protocol using over 180 SARS-CoV-2 positive samples to improve on the success rate of sequencing. Viral RNA was extracted from all samples using both QIAamp Viral RNA kit (Qiagen) and Quick-RNA Viral Kit (Zymoresearch). Sequencing of the genetic material was done using the Oxford nanopore MinION platform following an amplicon-based enrichment of the SARS-CoV-2 genome (ARTIC Protocol). In optimizing the starting material for derivation of quality amplicons for sequencing, the initial extraction and elution volumes were adjusted for both kits. This significantly improved the amplification success rate of the target SARs-CoV-2 genome. Comparison of SARS-CoV-2 genomes before and after optimization showed a significant improvement on the genome coverage with a high percentage of the genome having fewer gaps after sequence analysis which had previously precluded variant calling.

P41

Optimal Classification of Small Volumetric Dataset: 2D or 3D CNN in Infant Brain Age Prediction?* Mahdieh Shabanian1, Markus Wenzel2, and John P. DeVincenzo3 1Department of Biomedical Engineering, University of Tennessee Health Science Center, Memphis, TN, United States; 2Fraunhofer Institute for Digital Medicine MEVIS, Bremen, Germany; 3Enanta Pharmaceuticals, Watertown, MA, USA

Determining if the brain is developing normally is a key component of pediatric neuroradiology and neurology. Brain magnetic resonance imaging (MRI) of infants demonstrates a specific pattern of development beyond simply myelination. While radiologists have used myelination patterns, brain morphology and size characteristics to determine age-adequate brain maturity, this requires years of experience in pediatric neuroradiology. With no standardized criteria, visual estimation of the structural maturity of the brain from MRI before three years of age remains dominated by inter-observer and intra-observer variability. A more objective estimation of brain developmental age could help physicians identify many neurodevelopmental conditions and diseases earlier and more reliably. Such data, however, is naturally hard to obtain, and the observer ground truth not much of a gold standard due to subjectivity of assessment. In this light, we explore the general feasibility to tackle this task, and the utility of different approaches, including deep two- and threedimensional convolutional neural networks (CNN) that were trained on a fusion of T1-weighted, T2-weighted, and proton density (PD) weighted sequences from 84 individual subjects divided into four age groups from birth to 3 years of age. In the best performing approach, we achieved an accuracy of 0.90 [95% CI:0.86-0.94] using a 2D CNN on a central axial thick slab. We discuss the comparison to 3D networks and show how the performance compares to the use of only one sequence (T1w). In conclusion, despite the theoretical superiority of 3D CNN approaches, in limited-data situations, such approaches are inferior to simpler architectures. The code can be found in https://github.com/shabanian2018/Age_ MRI-Classification Keywords: Deep learning, CNN, neurodevelopmental, age estimation, infant diseases.

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P42

Modulation of SREBP-1c/PNPLA-3 pathway via PPAR-α with Synbiotic intervention in NAFLD model Dixa Sharma, Corresponding author: Dr. Palash Mandal Chaotar University of Science and Technology, India

Non-alcoholic fatty liver disease (NAFLD) is liver inflammation and a major threat to public health. Prediction about patient’s stratification and prognosis is difficult. This limits the rational basis for options of management. And thus, till date there is no Food and Drug Administration (FDA) approved drug to treat NAFLD. Genetic factors have prominent role in NAFLD. This study will analyse human serum samples from NAFLD patients to understand the genetic basis of human patatin-like protein 3 (PNPLA3). Evidences has provided the new insights into the NAFLD biology and underlined potential pharmaceutical targets. Synbiotic have antibacterial and anti- inflammatory properties, and may be potential candidates for NAFLD therapy. To study the molecular mechanism, HepG2 and Wistar rats were used to create NAFLD models and received treatments. Liver tissues were examined using HE stain, and serum biochemical indices were measured. The levels of peroxisome proliferators- activated receptor (PPAR)-α, sterol regulatory element binding protein-1c (SREBP1c), PNPLA-3, inflammatory cytokines and apoptosis biomarkers in liver tissues were measured by qRT-PCR. Tissue stain analysis indicates the hepatocyte with increased and huge lipid droplets in NAFLD models while synbiotic reduces the number and size of lipid droplets. Meanwhile, synbiotic decreased the liver markers levels and oxidative stress in serum. Synbiotic decreased the level of PPAR-α, SREBP-1c and PNPLA-3. Synbiotic reduces hepatic inflammation and apoptosis by affecting IL-6, TNF-α, caspase-3 and Bcl-2 in NAFLD models. Therefore, synbiotic may act as a therapeutic intervention for inhibiting the NAFLD progression by affecting SREBP- 1c/PNPLA-3 pathway via PPAR-α.

P43

Inflammatory Signatures Determines the fate of Murine Leptospirosis Advait Shetty1, Suman Kundu2, Maria Gomes-Solecki1,2 Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, Tennessee, U.S. Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, Tennessee, USA

Leptospirosis is a zoonotic infection caused by a spirochete called Leptospira. Rats are asymptomatic carriers of this bacteria, they can either directly or indirectly transmit the infection to other wild or domestic animals via contaminated urine. Humans are accidental host and can become infected through contaminated water from occupational exposure, natural disasters, recreational activities. Host-pathogen interaction during leptospirosis is a broad area of focus and needs in-depth investigation. Immunological consequences after pathogenic Leptospira evasion in the early stage is yet unexplored. Elimination of Leptospira by host depends on various factors, the key being early production of inflammatory markers like cytokines and chemokines, that activate specific immune cells, which eradicate the bacteria from the site of infection. In our study, we used a C3H/HeJ mice model, wherein we determined the early inflammatory response involved during pathogenic versus non-pathogenic Leptospira infection. Pathogenic L. interrogans resulted in production of major chemokines and cytokines at 24h and 72h post infection, engaging major immune cells in the spleen and resulted in splenomegaly. Thus, innate immune arsenals were not enough to combat with the pathogenic Leptospira and thereby led the engagement of adaptive immune arm. In contrary, non-pathogenic L. biflexa was mostly cleared out from the system and no dissemination occurred. The innate immune system is solely responsible in removal of non-pathogenic bacteria as indicated by our results. Our data suggests that differential signalling cascade of host immune system is the key decision maker to distinguish between pathogen versus non-pathogen which needs to be investigated.

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P44

Genomics and epigenetics guided identification of tissuespecific genomic safe harbors Dewan Shrestha, Aishee Bag, Yeting Zhang, Ruiqiong Wu, Xing Tang, Qian Qi, Jinchuan Xing, Yong Cheng University of Tennessee Health Science Center, Memphis, TN

Genomic safe harbors (GSHs) are regions of the genome that can maintain transgene expression without disrupting the function of host cells. GSHs play an increasingly important role in improving the efficiency and safety of genome engineering. However, limited GSHs have been identified. Here, we developed a framework to facilitate GSHs searches by integrating information from polymorphic mobile element insertions (pMEIs) that naturally occur in human populations, epigenomic signatures, and 3D chromatin organization. By applying our framework to pMEIs identified in the 1000 Genomes project and the Genotype-Tissue Expression (GTEx) project, we discovered 22 and 5 candidate GSHs in blood and brain cells, respectively. For one candidate blood GSH, we demonstrated the stable expression of transgene without transcriptome disruption in erythroid cells. We also developed a computer program for knowledge-based tissue specific GSH selection. Our study provides a new knowledge-based framework to identify tissue-specific GSHs. Combining with the fast-growing genome engineering technologies, our approach has the potential to improve the overall safety and efficiency of gene and cell-based therapy in the near future.

P45

Protective Effect of scaRNA20-modified Cardiomyocytes Exposed Under Hypoxic Condition* Vinoth Sigamani, Sheeja Rajasingh, Narasimman Gurusamy, Selene G. Perales, Johnson Rajasingh Integrated Biomedical Sciences, University of Tennessee Health Science Center

Introduction: The mRNA splicing is regulated by small Cajal-body associated RNAs (scaRNAs), which plays a significant role in mammalian cardiac development and differentiation. However, their potential contribution to human in-vitro differentiation of induced pluripotent stem cells (iPSCs) into induced cardiomyocytes (iCMCs) and its protective role remains unknown. In this study, we elucidated the role of scaRNA20- overexpressed iCMCs (scaRNA-OE-iCMCs) during hypoxic condition. Hypothesis: We hypothesize that scaRNA20 overexpressed iCMCs are capable of withstanding better under low oxygen environment by providing enhanced survival and anti-inflammatory response when compared to normal iCMCs. Methods & Results: Skin fibroblast-derived normal iCMCs (iCMCs) and scaRNA- OE-iCMCs were subjected to hypoxic condition (3%). Our novel contact-free particle image velocimetry (PIV) analysis of iCMCs showed that the contractility is reduced in normal iCMCs, whereas the scaRNA20-OE-iCMCs maintained the contractility under hypoxic condition. Furthermore, our qRT-PCR data revealed that scaRNA20-OE- iCMCs showed a significantly increased mRNA expression of cardiac-specific genes such as cardiac troponin T and GATA4 and at the same time showed a signiﬁcantly reduced expression of pro-inﬂamatory genes such as TNFa and IL11. Conclusion: These data suggest that the scaRNA20-OE-iCMCs are capable of withstanding the low-oxygen environment exist in the infarcted heart. We report for the first time, the beneficial effects of scaRNA20 modulated CMCs, and this modification prior to transplantation will provide better engraftment and effective therapy for cardiac regeneration.

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P46

Novel newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye Abijeet Singh Mehta, Anuradha Venkatakrishnan Chimata, Prajakta Deshpande, Panagiotis A Tsonis, Amit Singh University of Dayton, Dayton, OH

A fundamental process of regeneration, which varies among animals, recruits conserved signaling pathways to restore missing parts. Only a few animals like newts can repeatedly regenerate lost body parts throughout their lifespan due to strategic regulation of conserved signaling pathways by newt’s regeneration tool-kit genes. Lack of genetic tools pose challenges to determine the role of these genes. Here we report use of genetically tractable Drosophila eye model to demonstrate the potential of a group of unique protein(s) from newt (Notophthalmus viridescens), which when ectopically expressed can significantly rescue missing photoreceptor cells in a Drosophila L2 eye mutant. A mutant for Lobe (L) gene, L2, exhibits the loss of ventral half of early developing eye due to death of photoreceptor cells. We also tested their role during later stages of development by using gain-of-function of head involution defective (hid), an executioner caspase, when misexpressed using GMR-GAL4 (GMR>hid) triggers cell death resulting in a “no-eye” or reduced eye phenotype. Surprisingly ectopically expressing newt genes in L2 eye mutant backgrounds as well as GMR>hid background significantly restore missing photoreceptor cells. The phenotype restoration is due to upregulation of cell proliferation and blocking cell death. Additionally, these newt proteins with signal peptides motifs exhibit non-cell- autonomous rescue properties. In our study we also reported that Wingless (Wg)/Wnt is the evolutionarily conserved signaling pathway that is downregulated by these newt proteins to promote rescue of Drosophila eye mutants. Modulation of Wg/Wnt signaling levels by using effector or inhibitor of Wg/Wnt signaling pathway in eye mutant background where newt gene(s) is ectopically expressed suggests that Wg signaling acts downstream of newt genes. Our data highlights the restoration potential of novel newt proteins that regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells response in Drosophila model with weak restoration capability.

P47

Carbon “Quantum” Dots for Bioapplications Ya-Ping Sun Department of Chemistry, Clemson University, Clemson, South Carolina, USA

Carbon “quantum” dots or carbon dots (CDots) exploit and enhance the intrinsic photoexcited state properties and processes of small carbon nanoparticles via effective nanoparticle surface passivation by chemical functionalization with organic species. The optical properties and photoinduced redox characteristics of CDots are competitive to those of established conventional semiconductor quantum dots and also fullerenes and other carbon nanomaterials. Highlighted in this presentation are major advances in the exploration of CDots for their serving as high-performance yet nontoxic fluorescence probes for one- and multi-photon bioimaging in vitro and in vivo, their uniquely potent antimicrobial function to inactivate effectively and efficiently some of the toughest bacterial pathogens and viruses under visible/natural or ambient light conditions, and the their relevance and opportunities offered to applications in regenerative medicine.

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P48

Characterization of Activins βC and βE Signaling Profiles Kylie A. Vestal, Thomas B. Thompson University of Cincinnati, Cincinnati, OH

The Transforming Growth Factor β (TGFβ) superfamily is comprised of 33 independent proteins that play a major role in numerous biological processes, from driving developmental programs in the embryo to tissue regeneration and wound healing in the adult. The Activin proteins, including ActA, B, C, and E, are a subclass of proteins within the Transforming Growth Factor β (TGFβ) superfamily that have been well-characterized for their role in reproduction, inflammation, and cell proliferation. Ligands are disulfide-linked dimers formed from two β chains to generate either a homodimeric protein, such as ActA (InhβA:InhβA), or a heterodimeric protein, such as ActAB (InhβA:InhβB). While the functions of ActA and ActB have been well characterized, little is known about the biological role of ActC and ActE. Numerous studies have suggested that ActC and ActE are non-signaling inhibitors to ActA and ActB ligands. Our recent data has characterized the signaling of ActC and has identified the type I receptor, activin receptor-like kinase 7 (ALK7), as being essential to transmit an ActC signal. Because of sequence homology and recent work establishing the signaling profile of ActC, we predict that ActE may generate a SMAD2/3 response by activation of the activin receptor-like kinase 7 (ALK7) similar to ActC. While ActC and ActE are almost exclusively expressed in the liver, the discovery that ActC, and likely ActE, are signaling molecules raises the possibility that they function as newly discovered hepatokines.

P49

Disruption of Sonic Hedgehog Signaling impairs adult neurogenesis and leads to increased anxiety/depressionlike behavior in healthy and stroke animals* Jiapeng Wang1,2, Flavia Correa Turcato2, Alicia Bedolla1,3, Kierra Ware2, Maxwell Weed2, and Yu Luo2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati, Cincinnati, Ohio; Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio; Neuroscience Graduate Program, College of Medicine, University of Cincinnati, Cincinnati, Ohio

Sonic hedgehog (Shh) signaling has a nonredundant role in mediating developmental neurogenesis. However, the mechanisms underlying its effects on adult neurogenesis and cognitive behaviors under physiological or stroke conditions remain undefined. Our past study demonstrated administration of Shh receptor Smoothened (Smo) agonist in poststroke animals promotes adult neurogenesis and functional recovery in behavior. Here, by utilizing a neural stem cell (NSCs) specific Smo knockout (NSC-Smo cKO) mouse model, we investigated adult neurogenesis in young and aged Smo deficiency mice. Our results demonstrated a significant reduction of neurogenesis at both subventricular and hippocampal subgranular zone in 3month old NSC-Smo cKO mice. Interestingly, NSC-Smo cKO mice also showed an accelerated decline in neurogenesis through aging. Furthermore, behavior studies revealed that compromised neurogenesis in NSC- Smo cKO mice led to increased anxiety-like (Elevated Plus Maze) or depression-like (Forced Swim Test) behaviors, which supports the important role of adult neurogenesis in regulating anxiety behavioral outcomes. We also investigated whether Shh signaling contributes to ischemic injury-induced neurogenesis using the filament-based middle cerebral occlusion/ reperfusion (MCAO) model. Consistently, post-stroke animal behavior test also demonstrated ischemia- induced hypoxic damage increased animal anxiety level while disruption of Shh signaling aggravated the results in young-adult mice. In summary, our study demonstrated that the Shh signaling is essential for adult neurogenesis under both physiological and ischemic conditions. Our data also supported the important role of adult neurogenesis in modulating anxiety/depressionlike behavior in animals.

Keywords: Sonic hedgehog; Smo; Stroke; MCAO; Neurogenesis

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The Role of TGF-β signaling in adult neurogenesis* Kierra Ware College of Medicine, University of Cincinnati

Compromised adult neurogenesis in the subgranular zone (SGZ) of hippocampus is implicated in neurological disorders of the mammalian brain. Previous studies have demonstrated that microglia can modulate adult hippocampal neurogenesis, however, the exact molecular and cellular mechanisms is not well understood. TGF-β signaling has recently been indicated to be a key modulator for microglia homeostasis and TGF-β has also been implicated to be important in late stage maturation of neurons. Based on previous work and our preliminary data, we hypothesize that TGF-β signaling regulates adult neurogenesis through both a direct mechanism in adult NSCs and indirect mechanism through microglia-NSC crosstalk. To test this hypothesis, we have generated novel inducible NSC-or microglia-specific TGF-β receptor- TβRI(ALK5) knockout (KO) mice. Adult inducible specific silencing of TGF-β signaling in NSCs directly or in microglia cells indirectly will allow us to elucidate the precise role of TGF-β signaling on NSC activation, proliferation, and differentiation in addition to providing a platform to dissect out the crosstalk between microglia and neural stem cells in vivo. Our data show that microglia morphology shifts from a quiescent, ramified state to an activated phenotype in the absence of TGF-β1 and that in vivo ablation of microglia-derived transforming growth factor beta 1 (TGF-β1) ligand increases the number of adult-born immature neurons in the SGZ. To further elucidate the downstream molecular pathways of TGF-β signaling in different cell types (NSCs or microglia), we are currently carrying out FACS-RNAseq analysis. In addition, the behavioral outcome in these cell- type specific TGF-β cKO mice in regards to the adult neurogenesis-related functions (such as anxiety and cognitive flexibility) are also being analyzed.

P51

A Modified Chitosan-Polyethylene Glycol Bio-ink for use in Additive Manufacturing* Andrew Blass Watson, Joel D. Bumgardner, PhD, Tomoko Fujiwara, PhD University of Memphis

Chitosan is a cationic polysaccharide having two types of functional groups (-NH2 and -OH) that are capable of being selectively used for the addition of therapeutics and post-crosslinkers to achieve smart biofabrication. The 3D printing of chitosan hydrogels has attracted wide interest because of their excellent biocompatibility, biodegradability, and low cost. [1] Chitosan can be chemically modified with photo-polymerizable methacrylate (MA) groups, but chitosan scaffolds lack mechanical strength, limiting their use in tissue engineering. [1] Polyethylene glycol (PEG)-based hydrogels have proven extremely versatile for tissue engineering applications with superb mechanical strength. [2,3] We created a methacrylate modified chitosan (N-MAC) added difunctional polyethylene glycol dimethacrylate (PEGDMA) we created a doublenetwork gel with improved strength characteristics over that of chitosan hydrogels. To create the bio-ink, a 3 wt% solution of N-MAC is made with DI water 0.2 wt% of lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photoinitiator and 8 wt% PEGDMA. The N-MAC product was evaluated using Fourier-transform infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). To measure the curing time, a 365 nm wavelength light was used to irradiate bio-ink which was sandwiched between two glass slides. A hydrogel pattern can be visually observed forming, and the time for light irradiation was recorded as the curing time. The solution was cast into 13 mm tall, 9.7 mm diameter cylindrical test specimen for compression testing in an Instron mechanical tester. We were able to successfully print samples using this bio-ink using a Allevi bioprinter. Hydrogel cytocompatibility is currently being evaluated.

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P52

Modulation of GPCR Function by Membrane Lipids and Water* Nipuna Weerasinghe1, Steven D.E. Fried1, Anna R. Eitel1, Helen F. Mann1, Emily L. Cosgriff1, Andrey V. Struts1,2, Suchithranga M.D.C. Perera1, Michael F. Brown1 1University of Arizona; 2St. Petersburg State University

G-protein-coupled receptors (GPCRs) are integral membrane proteins that regulate a wide range of normal and diseaserelated physiological processes. Our understanding of the role of soft matter such as membrane lipids and cellular water on activation and regulation of the GPCR function remains incomplete. In the standard biochemical model, GPCRs behave as primarily agonist dependent bimodal switches, with little influence of the surrounding medium. However, using the visual receptor rhodopsin as a model GPCR, we show that water drives rhodopsin to a partially disordered, solvent-swollen conformational ensemble upon light absorption, rendering the standard model obsolete [1], [2]. We placed rhodopsin under varying degrees of osmotic stress using polyethylene glycol solutes and investigated the activation equilibrium response using UV-Visible spectroscopy. Our results show a flood of ~ 80 water molecules into the rhodopsin interior during photoactivation, a result supported by atomistic molecular dynamics simulations [3]. Furthermore, the osmolyte effects on rhodopsin activation are size dependent: large osmolytes back shift the equilibrium to inactive metarhodopsin-I (MI), while small osmolytes forward-shift the equilibrium to active metarhodopsin- II (MII). We attribute these size effects to varying degrees of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation of rhodopsin. Besides osmotic pressure, the application of hydrostatic pressure also back shifts the metarhodopsin equilibrium but for fundamentally different reasons. Integrating the two force-based methods together with neutron scattering experiments indicates that the active state of rhodopsin is more hydrated yet simultaneously more structurally collapsed [4]. At the same time, the active GPCR undergoes dynamic volume fluctuations and solvent coupling, which give rise to greater thermal volume. Our results necessitate a new understanding of GPCR activation in which the surrounding soft matter is paramount in governing conformational energy landscapes.

[1] N. Weerasinghe et al. (2018) Biophys. J., 114, 274a [2] N. Weerasinghe et al. (2018) FASEB J., 32, lb64 [3] N. Leioatts et al. (2014) Biochemistry 53, 376−385 [4] S.M.D.C Perera et al. (2018) J.Phys.Chem.Lett. 9, 7064–7071

P53

Characterization of astrocyte profile following microglia ablation in the acute and subacute MPTP mouse models* Elliot R. Wegman Molecular Genetics, University of Cincinnati

Inflammation and glial cell activation are one of the key characteristics of Parkinson’s disease (PD) pathology; however, their role in disease progression is not clear. Previous studies have implicated microglia can activate A1 neurotoxic astrocytes, which can act as a potential mediator of dopaminergic neuron cell death in animal PD models. Using the CSF-1R inhibitor PLX5622 to ablate microglia, we have demonstrated differential roles of microglia and astrocytes between the acute and subacute MPTP mouse models of PD. Previous studies have reported different modes of dopaminergic cell death between these two models: apoptosis in the subacute MPTP and necrosis in the acute MPTP model. Our results show ablation of microglia in the subacute MPTP model is neuroprotective, while ablation of microglia in the acute MPTP model is detrimental. Interestingly, in both models, ablation of microglia is accompanied by increased activation of astrocytes. Based on these results, we hypothesize that the different responses between these two models to microglia ablation stem from a neuroprotective astrocyte profile in the subacute MPTP model, and a neurotoxic astrocyte profile in the acute MPTP model. To test this hypothesis, we examined A1 vs. A2 markers in these two models with or without microglia using RNA-scope and IHC. Additionally, we will use RNA-seq to analyze the gene expression profile of FACS-sorted astrocytes following microglia ablation in these two models. Our data will provide insight into the molecular pathways regulating microglia and astrocyte crosstalk, and identify novel markers defining neuroprotective vs. neurotoxic astrocyte profiles in PD models.

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P54

Elevated bile acids compensate for Car deletion-induced impairment in liver regeneration after partial hepatectomy Weinan Zhou1, Ullas V Chembazhi2, Sushant Bangaru2, Angela E. Dean3, Jiansheng Huang4, David A. Rudnick4, 5, Auinash Kalsotra2, and Sayeepriyadarshini Anakk1* 1Department of Molecular and Integrative Physiology, University of Illinois at Urbana- Champaign, Urbana, IL; 2Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL; 3Division of Nutritional Sciences, University of Illinois Urbana Champaign, Urbana, IL; 4Department of Pediatrics, St. Louis Children's Hospital, Washington University School of Medicine, St. Louis, MO; 5Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO

*Corresponding author (Email: anakk@illinois.edu)

The liver functions as a central hub for nutrient and xenobiotics metabolism, thus making it susceptible to injury. The liver maintains regenerative ability and coordinates metabolism with proliferation to ensure proper function. Constitutive androstane receptor (CAR) transcriptionally regulates bile acid (BA) detoxification and hepatocyte proliferation. But, how CAR coordinates proliferation and BA homeostasis during regeneration remains poorly understood. We examined the liver weight, hepatocyte proliferation, and BA levels in male and female Car knockout (CarKO) and wild-type (WT) mice at different time-points after 2/3rd partial hepatectomy (PHx). We also repeated these experiments in the presence of BA sequestrant cholestyramine (CHR). Compared to WT, CarKO mice exhibited similar liver weight and hepatocyte proliferation except for a dramatic increase in hepatic and systemic BAs after PHx. Car deficiency altered the expression of genes involved in proliferation and BA metabolism in a sex-specific manner. CHR treatment, as expected, reduced the circulating BAs in both WT and CarKO mice. Intriguingly, CHR-fed CarKO mice displayed reduced liver weight compared to WT mice at 36 hours after PHx, indicating a potential deficit in liver regeneration in CarKO mice in the absence of excess BAs. Finally, we analyzed previously published single-cell RNA seq data of regenerating mouse livers and found that Car and its targets are more highly expressed in hypermetabolic hepatocytes than proliferating hepatocytes. Overall, our results show that CAR plays a critical role in regulating and integrating BA metabolism with hepatocyte proliferation during liver regeneration.

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