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We believe science is essential for all Nebraskans 
Biomedical Sciences
BIOMEDICAL SCIENCES Chairperson: Dr. Annemarie Shibata
Moderators(s): Drs. Paul Denton, Joseph Dolence, Ian Hall, James Fletcher, Kimberly A. Carlson, Leslie Rault-Bucklin, Lynne Dieckman, and Gwendalyn King
FRIDAY, APRIL 19
MORNING SESSION - A1 Location: Great Plains Room A
Moderator: Dr. Paul Denton
7:30 Presenters upload Session 1 talks from USB drives or cloud onto the room computer.
7:45 Session opens for participants to join ZOOM
8:00 STAPHYLOCOCCUS AUREUS PERSISTER CELLS EXHIBIT HIGHER TOLERANCE TO INNATE IMMUNE COMPONENTS. Kenan Brodd, Emma Weis, Alexis Hobbs, Kimberly A. Carlson, and Austin Nuxoll (abstract)
8:15 NK CELL PHENOTYPING USING TWO COMPLEMENTARY FLOW CYTOMETRIC ANALYSIS STRATEGIES. Isabelle S. Weber, Maia M.C. Bennett-Boehm, and Paul W. Denton (abstract)
8:30 SURFACE CD16 MODULATION BY TOLL-LIKE RECEPTOR 9 (TLR9) AGONISM ON HUMAN NATURAL KILLER CELLS. Angela N. Truong, Anna R. Mahr, Maia M.C. Bennett-Boehm, and Paul W. Denton (abstract)
8:45 DEVELOPMENT OF COMPUTATIONALLY DESIGNED, ADNOVIRAL-VECTORED INFLUENZA A VACCINES TARGETING NEURAMINIDASE. Amber Gadeken, Lauren Sturgell, Matt Pekarek, and Dr. Eric Weaver (abstract)
9:00 INVESTIGATION OF MAMMALIAN IFITM3 GENES AS VIRAL RESTRICTION FACTORS OF HSV-1 INFECTION. Hayden Klaus and Dane Bowder (abstract)
9:15 KNOCKING OUT THE IMMUNITY REPRESSOR GENE IN THE PHAGE JABITH AND THE INTEGRASE GENE IN THE PHAGE LILHOMIEP HAS THE POTENTIAL TO CHANGE THE PHAGE LIFE CYCLE. Tiffany Carnahan and Erin Doyle (abstract)
9:30 BREAK
During break - Presenters upload Session 2 talks from USB drives or cloud onto the room computer.
MORNING SESSION - B1 Location: Great Plains Room B
Moderator: Dr. Ian Hall
7:30 Presenters upload Session 1 talks from USB drives or cloud onto the room computer.
7:45 ZOOM Session opens for participants to join ZOOM
8:00 STRUCTURAL STUDIES OF A EUKARYOTIC OAZ1-PK RNA. Andree Kakish, Lindsay Nedungadi, Zach Frevert, Lucas Struble, Gloria Borgstahl, and Juliane Soukup (abstract)
8:15 OPTOELECTROCHEMICAL METHOD FOR READING DNA MICROARRAY ON AN AU MONOLITH ELECTRODE. Keegan Nitsch, Ravi F. Saraf, Akshat Saraf, Jay Min Lim, Joydip Dey, and Khirul Alam (abstract)
8:30 STRUCTURAL AND DYNAMIC STUDIES OF THE INTERACTION BETWEEN TWO PROTEINS INVOLVED IN GENE-SILENCING. Emily Ekstrum, Evan Veltri, Molly Dolan, Zachary Frevert, and Lynne Dieckman (abstract)
8:45 SINGLE MOLECULE STUDIES OF PCNA AND CAF-1 USING TIRF MICROSCOPY. Olivia Nicholson, and Dr. Lynne Dieckman (abstract)
9:00 EXAMINING THE EFFECTS OF TRADITIONAL CULTURING TECHNIQUES ON FILAMENTATION ASSAYS IN THE FUNGUS CANDIDA ALBICANS. Ethan Funke and Jill Blankenship (abstract)
9:15 INVESTIGATING THE ROLE FOR DIACYLGLYCEROL IN HEAT TOLERANCE IN DROSOPHILA MELANOGASTER. Sunayn Cheku, Blase Rokusek, Sunanda Rajput, Lawrence Harshman, and Kimberly A. Carlson (abstract)
9:30 BREAK
During break - Presenters upload Session 2 talks from USB drives or cloud onto the room computer.
MORNING SESSION - C1 Location: Great Plains Room C
Moderator: Dr. Joseph Dolence
7:30 Presenters upload Session 1 talks from USB drives or cloud onto the room computer.
7:45 Session opens for participants to join ZOOM
8:00 THE ROLE OF SERUM ALBUMIN IN FILAMENTATION AND FARNESO SEQUESTRATION. Brigid M. Toomey, Daniel J. Gutzmann, Kenneth W. Nickerson, and Audrey L. Atkin (abstract)
8:15 INVESTIGATING THE POTENTIAL SURVIVAL ADVANTAGE OF STAPHYLOCOCCUS AUREUS PERSISTERS WITHIN A MACROPHAGE ENVIRONMENT. Carter Moss, Emma Weis, Kenan Brodd, Alexis Hobbs, Kimberly A. Carlson, and Austin Nuxoll (abstract)
8:30 SEX DIFFERENCES IN INNATE IMMUNE RESPONSE FOLLOWING PEANUT EXPOSURE. Sunanda Rajput and Joseph Dolence (abstract)
8:45 EXPLORING MOMP AS A TARGET FOR AN mRNA CHLAMYDIA VACCINE. Parker Tinsley, Shawn Pearcy and Douglas Christensen (abstract)
9:00 INVESTIGATING THE ROLE OF LINCRNA NOSTRILL IN ANTIVIRAL AND INFLAMMATORY RESPONSES IN NEURONS. Aaron Marta, Sophie Ciechanowski, Jodi Hallgren, Hannah Pflum, Paige Harty, Davis Thalhuber, Kirsten Drescher, and Annemarie Shibata (abstract)
9:15 INVOLVEMENT OF PUTATIVE MEMBRANE RAFT PROTEINS IN CAENORHABDITIS ELEGANS’ INNATE IMMUNE RESPONSE TO PATHOGENIC STENOTROPHOMONAS MALTOPHILIA. Minjae Song and Michael Herman (abstract)
9:30 BREAK
During break - Presenters upload Session 2 talks from USB drives or cloud onto the room computer.
MORNING SESSION - D1 Location: Garden Room
Moderator: Dr. James Fletcher
7:30 Presenters upload Session 1 talks from USB drives or cloud onto the room computer.
7:45 Session opens for participants to join ZOOM
8:00 SCREENING OF PROMISING DRUG-LIKE COMPOUNDS FOR ACTIVITY AGAINST PARASITIC AMOEBA, NAEGLERIA. Brianna N. Davis, Andrew J. Neville, Jeff M. Zimmerly, and Paul H. Davis (abstract)
8:15 ISOLATION OF CERE FROM WILLAERTIA MAGNA AND TETRAMITUS THORNTONI. Isaac Richards (abstract)
8:30 PEPTIDE AMPHIPHILES AS A NOVEL DRUG DELIVERY SYSTEM. Andrew Wegner, Martin Conda-Sheridan, Huihua Xing, and Jiachen Feng (abstract)
8:45 DIFFERENTIAL INTERACTIONS WITHIN THE GUT MICROBIOME OF MARMOSETS TREATED WITH ANTIBIOTICS AS REVEALED BY COMPUTATIONAL NETWORK ANALYSIS. Jordan B. Hernandez, Shivdeep S. Hayer, Sophie Alvarez, Kathryn Cooper, Mahsa Mohammadi, Hyun-Seob Song, and Jonathan B. Clayton (abstract)
9:00 COMBATING FOSFOMYCIN RESISTANCE VIA INHIBITION OF FOSB WITH GLCNAC-MAL. David Johnson and Mary E. Keithly (abstract)
9:15 EFFICIENCY OF TOOTH WHITENING PRODUCTS AND THE EFFECT ON THE TOOTH. Ashley Christianson and Mary Keithly (abstract)
9:30 BREAK
During break - Presenters upload Session 2 talks from USB drives or cloud onto the room computer.
MORNING SESSION - A2 Location: Great Plains Room A
Moderator: Dr. Paul Denton
9:40 Session opens for participants to join ZOOM
9:45 ANALYZING KILLING FUNCTIONS OF HUMAN NATURAL KILLER CELLS THROUGH A TRANSCRIPTIONAL LENS VIA SINGLE-CELL RNA SEQUENCING. Cami R. Bisson and Paul W. Denton (abstract)
10:00 SPANNING PARADIGMS: USING IMMUNOTHERAPY TO ENHANCE HUMAN NATURAL KILLER CELL ANTIBODY DEPENDENT CELL-MEDIATED CYTOTOXICITY AGAINST CANCER AND INFECTIOUS DISEASE. Jaden Nienhueser and Paul W. Denton (abstract)
10:15 DEVELOPMENT OF A NOVEL ASSAY TO ASSESS MACROPHAGE PHENOTYPE. Furqan Mahdi, Kayla Ney, and Rebecca Wachs (abstract)
10:30 SNIFFING OUT PROINFLAMMATORY FACTORS – MODULATION OF MICROGLIAL INFLAMMATORY RESPONSE THROUGH NOVEL LONG NON-CODING RNA. Sophie Ciechanowski, Aaron Marta, Olivia Burleigh, Nicholas Mathy, Kristen Drescher, and Annemarie Shibata (abstract)
10:45 NOSTRILL MEDIATED BEHAVIORAL DIFFERENCES FOLLOWING ACUTE AND CHRONIC INDUCED DEMYELINATING DISEASE PATHOLOGIES. Hannah Pflum, Jodi Hallgren, Sophie Ciechanowski, Aaron Marta, Paige Harty, Kristen Drescher, and Annemarie Shibata (abstract)
MORNING SESSION – B2 Location: Great Plains Room B
Moderator: Dr. Ian Hall
9:40 ZOOM Session opens for participants to join ZOOM
9:45 NOVEL INTERACTION BETWEEN TWO PROTEINS INVOLVED IN NUCLEOSOME ASSEMBLY. Grace Jaworski and Lynne Dieckman (abstract)
10:00 CHARACTERIZATION OF THE NUCLEAR LOCALIZATION SIGNAL (NLS) OF ORF1 OF NORA VIRUS. Belle Turk, Amanda J. Macke, Darby J. Carlson, Alexis M. Hobbs, and Kimberly A. Carlson (abstract)
10:15 SHAPE ANALYSIS OF CRASSOTREA GIGAS OAZ-PK RNA. Elizabeth Alberts and Juliane Strauss-Soukup (abstract)
10:30 CHARACTERIZING HIGH PERSISTER PHENOTYPES IN STAPHYLOCOCCUS EPIDERMIDIS CLINICAL ISOLATES. Mariam Garcia, Kaitlyn Pineda, Kael Kingery, Alexis Hobbs, Kimberly A. Carlson, and Austin Nuxoll (abstract)
10:45 USING 3D PRINTED DEVICES TO ELUTE AND CONCENTRATE S. CEREVISIAE DNA. Esmeralda Mendez, Thi Huynh, and Kristy Kounovsky-Shafer (abstract)
MORNING SESSION – C2 Location: Great Plains Room C
Moderator: Dr. Joseph Dolence
9:40 Session opens for participants to join ZOOM
9:45 STRUCTURAL ANALYSIS OF OAZ RNA IN NEUROSPORA CRASSA. Caitlin Sousley, Emma Curran, and Juliane Strauss-Soukup (abstract)
10:00 MUCIN 5AC MODULATES CANCER-ASSOCIATED FIBROBLAST HETEROGENEITY THROUGH EPIGENETIC REGULATION. Rachel Kehrberg, Xiaoqi Li, Surinder K. Batra, and Sushil Kumar (abstract)
10:15 HISTOLOGICAL VALIDATION OF NADH FLIM ANALYSIS TO NON-INVASIVELY DETECT SKIN CANCER. Jonathan Li, Alex Chen, Kennedy A. Haase , Jackson M. Laurent, Maimuna Olow Nagey, Derek A.Remitar III, Abraham J. Saks, Hannah Schloman, Jinann A. Shoshara, Zachary J. Smith, Fiona Sun, Jacob A. Sweet, Jake S. Wakahir, Michael Nichols, and Laura Hansen (abstract)
10:30 EFFECT OF CURCUMIN ON EXPRESSION OF GROWTH AND SURVIVAL GENES IN TRIPLE NEGATIVE BREAST CANCER. Emmanuella Tchona, Isioma Akwanamnye, and Ann Marie Buchmann (abstract)
10:45 INVESTIGATING THE ROLE OF HCMV US18 IN VIRAL INFECTION. Mary Helms, Douglas Christensen, and Lindsey B. Crawford (abstract)
MORNING SESSION – D2 Location: Garden Room
Moderator: Dr. James Fletcher
9:40 Session opens for participants to join ZOOM
9:45 UTILIZING A COMPREHENSIVE BACTERIAL SCREENING PANEL TO IDENTIFY NOVEL ANTIMICROBIAL COMPOUNDS. Jeff Zimmerly, Nicholas Armstrong, Clare Euteneuer, Yunos Aliza, and Paul H. Davis (abstract)
10:00 COMBATING ANTI-MICROBIAL RESISTANCE UTILIZING OPENTRONS TECHNOLOGY. Nicholas Armstrong, Jeffrey Zimmerly, Yunos Alizai, and Paul H. Davis (abstract)
10:15 EXPLORING THE ROLE OF INLB FOR USE AS A DRUG DELIVERY AGENT. Jason Franklin, Shawn Pearcy Ph.D. , and Douglas Christensen Ph.D. (abstract)
10:30 POLYVALENT PYRIDINIUM- AND QUINOLINIUM-SUBSTITUTED TRIAZOLES AS ANTISEPTICS. Adam Burr and James T. Fletcher (abstract)
10:45 DENTAL CARIE RESISTANCE AND THE APPLICATION OF SEALANTS. Jamie Graff and Rachelle Rider (abstract)
11:00 MAIBEN LECTURE Location: Great Plains Room B-C
Mr. Grant Reiner, “Platte Basin Timelapse: A Watershed in Motion”
11:45 SCHOLARSHIPS AND AWARDS Location: Great Plains Room B-C
12:00 STATE OF THE ACADEMY Location: Great Plains Room B-C
12:15 LUNCH Location: East Union Cafeteria
12:15 INBRE Steering Committee Meeting Location: Arbor Suite A
AFTERNOON SESSION - A3 Location: Great Plains Room A
Moderator: Dr. Leslie Rault-Bucklin
1:15 Presenters upload Session 3 talks from USB drives or cloud onto the room computer.
1:25 Session is open for participants to join ZOOM
1:30 ABC TRANSPORTER MODULATING RESPONSES TO LLINS FOR MALARIA
MOSQUITOES. Heather M. Hernandez, Leslie C. Rault, and Troy D. Anderson (abstract)
1:45 PHAGOSTIMULANT ACTIVITY OF AMINO ACIDS TO IMPROVE NEXT-GENERATION
ATTRACT-AND-KILL TECHNOLOGIES. Xi Xian Ng, Ellis J. Johnson, Leslie C. Rault, and Troy D. Anderson (abstract)
2:00 IMPROVING AN OPEN-SOURCED AUTOMATED MICROPLATE ASSAY FOR ANTI-INFECTIVE AND CYTOTOXIC COMPOUND SCREENING. M. Yunos Alizai, Brianna N. Davis, and Paul H. Davis (abstract)
2:15 IDENTIFICATION OF STAPHYLOCOCCUS LUGDUNESIS PROTEASES THROUGH ETHYL METHANESULFONATE MUTAGENESIS. Caleb Rother, Justine Pitzer, and Austin Nuxoll (abstract)
2:30 WURST PROTEIN PLAYS A CRUCIAL ROLE IN THE MOLTING PROCESS OF INSECTS. Savannah Armendariz, Faith Kozisek, Jonathan Cenovic, and Sujata S Chaudhari (abstract)
2:45 BREAK
During break - Presenters upload Session 4 talks from USB drives or cloud onto the room computer.
AFTERNOON SESSION - B3 Location: Great Plains Room B
Moderator: Dr. Kimberly A. Carlson
1:15 Presenters upload Session 3 talks from USB drives or cloud onto the room computer.
1:25 Session is open for participants to join ZOOM
1:30 QUANTIFICATION OF NADH PHASOR FLIM AND P53 EXPRESSION FOR UV-INDUCED SKIN CANCER IN VIVO. Greer L. Porter, Alex Chen, Kennedy A. Haase, Reese Kolar, Jackson M. Laurent, Johnathan Li, Aidan O’ Mara, Maimuna Olow Nagey,Jalen K. Ramos, Derek A. Remitar III, Abraham J. Saks, Hannah Schloman, Jinann A. Shoshara, Zachary J. Smith, Fiona Sun, Jacob A. Sweet, Jake S. Wakahiro, Laura A. Hansen, Michael G. Nichols (abstract)
1:45 INVESTIGATING METABOLIC CHANGES UNDER NORMOXIC AND HYPOXIC CONDITIONS THROUGH NADH PHASOR FLIM IN VITRO WITH INTEREST IN DEVELOPMENT OF A NON-INVASIVE DIAGNOSTIC BIOPSY IN VIVO. Abraham J. Saks, Jackson M. Laurent, Elizabeth M. Cronin, Greer L. Porter, Derek A. Remitar, Aidan J. O’Mara, Jinann A. Shoshara, Reese A. Kolar, Kennedy A. Haase, Laura A. Hansen, Michael G. Nichols (abstract)
2:00 REGULATION OF VLDLR GENE EXPRESSION BY MICRO RNA-100 IN MCF-7 HUMAN BREAST CANCER CELLS BEFORE AND AFTER THE EPITHELIAL TO MESENCHYMAL TRANSITION. McKenna Revis and Kate Marley (abstract)
2:15 EFFECT OF HIGH GLUCOSE ON FOCAL ADHESION AND RHO KINASE IN BREAST CANCER CELLS. Payton Sindelar and Surabhi Chandra (abstract)
2:30 AGGRESSIVE PROLIFERATION IN ADVANCED PROSTATE CANCER. Kaitlin Smith, Avery Stillahn, Sreyashi Bhattacharya, Samikshan Dutta, and Kaustubh Datta (abstract)
2:45 BREAK
Presenters upload Session 4 talks from USB drives or cloud onto the room computer.
AFTERNOON SESSION – C3 Location: Great Plains Room C
Moderator: Dr. Lynne Dieckman
1:15 Presenters upload Session 3 talks from USB drives or cloud onto the room computer.
1:25 Session is open for participants to join ZOOM
1:30 EXPLORING THE INTERACTION OF HCMV UL5 WITH HOST CELLULAR GENES ANKRD13A AND ST3GAL1. Kai Waddell, Kamryn Pfenning, and Lindsey Crawford (abstract)
1:45 THE PURSUIT OF PYRUVATE DEHYDROGENASE KINASE INHIBITORS TO TREAT METABOLIC DISEASE. Noah Shackelford, Allen A. Thomas and Michael A. Moxley (abstract)
2:00 CHARACTERIZING AN ESSENTIAL AMINO ACID REQUIRED FOR NUCLEOSOME ASSEMBLY. Emma Foley and Lynne Dieckman (abstract)
2:15 EVALUATION OF RESIDUAL PRIONS FROM DISINFECTED PRION-CONTAMINATED SURFACES UTILIZING RT-QUIC. Vivianne Payne and Jason Bartz (abstract)
2:30 EXAMINING SEX DIFFERENCES IN THE RESPONSE OF LUNG TYPE 2 INNATE LYMPHOID CELLS TO PEANUT. Alethia Henderson, Leigh-Anne Lehmann, Sunanda Rajput, Nicholas J. Hobbs, Ph.D., and Joseph J. Dolence (abstract)
2:45 BREAK
During break - Presenters upload Session 4 talks from USB drives or cloud onto the room computer.
AFTERNOON SESSION – D3 Location: Garden Room
Moderator: Dr. Gwendalyn King
1:15 Presenters upload Session 3 talks from USB drives or cloud onto the room computer.
1:25 Session is open for participants to join ZOOM
1:30 EXPLORING CHANGES IN COACTIVATION, STRENGTH, AND GROSS DEXTERITY BEFORE AND AFTER AN 8-WEEK HOME INTERVENTION IN CHILDREN WITH UPPER LIMB REDUCTIONS. Liliana Delgado and Dr. Jorge Zuniga (abstract)
1:45 POST-TRAUMATIC STRESS DISORDER SEVERITY FOLLOWING A TRAUMATIC EVENT MODULATION BY NEIGHBORHOOD-LEVEL FACTORS. Henry Drvol and Trey Andrews (abstract)
2:00 COMMUNICATION ERROR: RELATIONSHIP BETWEEN NEURAL CELL ADHESION MOLECULE EXPRESSION AND MUSCLE ACTIVATION IN PARKINSON’S DISEASE. Alexis Tonnemacher, Mitchel Magrini, and Kelley Hammond (abstract)
2:15 THE POTENTIAL EFFECTS OF AN FDA-APPROVED ANTIDEPRESSANT TRAZODONE ON DYSLIPIDEMIA. Naara Ramirez and Yipeng Sui (abstract)
2:30 THE EFFECT OF PROPRIOCEPTIVE NEUROMUSCULAR FACILITATION ON BALANCE. Kayla Brown and Rachelle Rider (abstract)
2:45 BREAK
During break - Presenters upload Session 4 talks from USB drives or cloud onto the room computer.
AFTERNOON SESSION – A4 Location: Great Plains Room A
Moderator: Dr. Leslie Rault-Bucklin
2:55 Session is open for participants to join ZOOM
3:00 EXPLORING THE INFLAMMATION REGULATORY FUNCTION OF LONG NON-CODING RNA EXPRESSION IN AMINOGLYCOSIDE INDUCED OTOTOXICITY. Collin Jackson, Emily Daffer, Tian Cong, Peter Steyger, and Annemarie Shibata (abstract)
3:15 DETERMINING EXPRESSION OF LNCRNAS IN AMINOGLYCOSIDE INDUCED OTOTOXICITY. Emily Daffer, Collin Jackson, Tian Cong, Peter Steyger, and Annemarie Shibata (abstract)
3:30 DEVELOPMENT OF VAPING PEANUT ALLERGY MOUSE MODELS. Marissa Hoover, Joseph Roeder, Zane Carlson, and Joseph J. Dolence, Ph.D. (abstract)
3:45 EXAMINING HOW SEX DIFFERENCES INFLUENCE THE ABILITY OF NEUTROPHILS AND DENDRITIC CELLS TO MOUNT RESPONSE FOLLOWING INHALATION OF PEANUT. Dana Dubas, Tyler Shaner, Sunanda Rajpu, McKenna S. Vininski, Nicholas J. Hobbs, and Joseph J. Dolence (abstract)
4:00 BREAK
END OF ORAL TALKS PROCEED TO THE POSTER SESSION
Session Chairs end afternoon sessions and Poster Presenters set up posters in the Great Plains Room B-C.
AFTERNOON SESSION – B4 Location: Great Plains Room B
Moderator: Dr. Kimberly A. Carlson
2:55 Session is open for participants to join ZOOM
3:00 ANALYSIS OF THE MICROBIOME OF ORNATE BOX TURTLES. Blake Skoumal, Victoria Martinez, Franziska C. Sandmeier, and Erin Doyle (abstract)
3:15 MEASUREMENT OF BINDING CONSTANTS FOR FASCIOLA HEPATICA FATTY ACID BINDING PROTEIN (FH-V) AND VARIOUS HYDROPHOBIC LIGANDS. Frank A. Kovacs and Mahesh Pattabiraman (abstract)
3:30 ACCESSING GERM TUBE FORMATION AND FARNESOL RESPONSE IN CANDIDA ALBICANS. Shyanne Urbin, Daniel J. Gutzmann, Kenneth Nickerson, and Audrey Atkin (abstract)
3:45 CHARACTERIZATION OF GUSTATORY RECEPTORS FROM WESTERN CORN ROOTWORM. Hope Hixson and Dr. Hideaki Moriyama (abstract)
4:00 BREAK
END OF ORAL TALKS PROCEED TO THE POSTER SESSION
Session Chairs end afternoon sessions and Poster Presenters set up posters in the Great Plains Room B-C.
AFTERNOON SESSION – C4 Location: Great Plains Room C
Moderator: Dr. Lynne Dieckman
2:55 Session is open for participants to join ZOOM
3:00 KINETIC STUDIES OF PROTEINS INVOLVED IN GENE SILENCING USING SURFACE PLASMON RESONANCE. Ivy Williams and Lynne Dieckman (abstract)
3:15 STRUCTURAL ANALYSIS OF CRASSOSTREA GIGAS OAZ-PK RNA. Hannah Ladwig, Rhiannon McCracken, and Juliane Soukup (abstract)
3:30 INVESTIGATING THE FUNCTION OF THE TRANSCRIPTION ELONGATION FACTOR, GREA, IN CHLAMYDIA TRACHOMATIS GROWTH AND DEVELOPMENT. Abigail Swoboda and Scot Ouellette (abstract)
3:45 USING BACTERIOPHAGES TO TREAT PLANT INFECTIONS OF P. SYRINGAE. Sierra Laschanzky, Katelyn Jindra, and Erin Doyle (abstract)
4:00 ENAMEL EROSION DUE TO ENERGY DRINKS. Jaylee Shaw and Dr. Mary Keithly (abstract)
END OF ORAL TALKS PROCEED TO THE POSTER SESSION
Session Chairs end afternoon sessions and Poster Presenters set up posters in the Great Plains Room B-C.
AFTERNOON SESSION – D4 Location: Garden Room
Moderator: Dr. Gwendalyn King
2:55 Session is open for participants to join ZOOM
3:00 TOWARDS A MATHEMATICAL UNDERSTANDING OF PRION STRAIN DYNAMICS. Josh Gilbert, Clay Cressler, and Jason C. Bartz (abstract)
3:15 SELECTIVE INTERACTIONS OF MAMMALIAN PRION PROTEINS TO THE SURFACE OF LIPID MEMBRANES: A COARSE- GRAINED MOLECULAR DYNAMICS SIMULATIONS STUDY. Noah Greenwood, Jamie Janos, Mason Borgman, and Patricia Soto (abstract)
3:30 INVESTIGATING ANXIOLYTIC RESPONSES OF DANIO RERIO THROUGH THE ROLE OF N-ACETYLCYSTEINE AMIDE ON THE GLUTAMATERGIC NEUROTRANSMITTER PATHWAY. Brooklynn K. Schmidt and Ryan Wong (abstract)
3:45 3D TISSUE MORPHOMETRY FOR NPRT AGAINST BRAIN TUMORS. Joshua Taylor, Dylan Bui, Jordyn Rockwell, and Andrew Ekpenyong (abstract)
4:00 BREAK
END OF ORAL TALKS PROCEED TO THE POSTER SESSION
Session Chairs end afternoon sessions and Poster Presenters set up posters in the Great Plains Room B-C.
4:15-5:15 POSTER SESSION Location: Great Plains Room B-C
Poster presenters please be available to present your poster during the hour presentation time.
4:15-4:45 BUSINESS MEETING Location: Great Plains Room A
PEP, EC, and LRP Committee Meeting
4:30-6:30 SOCIAL EVENT Location: Great Plains Room B-C
NE-INBRE-sponsored event. Announcement of Raffle Winners (must be present to win). Attend Posters, meet with colleagues, make connections, socialize. Hors d'oeuvres and soft drinks are provided. Additional cash bar.
POSTER SESSION Location: Great Plains Room B-C
4:15-5:15 Poster presenters please be available to present your poster during the hour presentation time.
BMS-1 EXPLORING THE INTERSECTION BETWEEN CHLAMYDIA TRACHOMATIS IRON STARVATION RESPONSE AND PATHOGEN RECOGNITION BY THE HOST. Monisha Alla, Nick Pokorzynski, and Rey Carabeo (abstract)
BMS-2 A MATHEMATICAL MODELING APPROACH TO EVALUATE THE BIOLOGICAL SIGNIFICANCE OF A NOVEL MEANS OF TRP ATTENUATION IN CHLAMYDIA TRACHOMATIS. Tyler Zimmerman, Nick Pokorzynski, and Rey Carabeo (abstract)
BMS-3 THE IMPACT OF MICROBIOME IN THE ENRICHMENT OF CANCER STEM CELLS AND PROGRESSION OF PANCREATIC CANCER. Kirtana Arikath, Prabagaran Narayanasamy, Seoung-Ryoung Choi, Zahraa Wajih, Alsafwani, Surinder.K.Batra, and Moorthy P. Ponnusamy (abstract)
BMS-4 MOLECULAR CONTROL OF TUMOR INITIATION IN COLORECTAL CANCER. Sarah Dharmaji, Heidi Vieira, Dianna Huisman, Deepan Chatterjee, James Askew, and Robert Lewis (abstract)
BMS-5 IDENTIFICATION OF NOVEL METABOLIC SIGNATURE IN PANCREATIC CANCER STEM CELLS. Neelanjana Gayen, Nivedeta Krishna Kumar, Ayoola O. Ogunleye, Karthik Balakrishnan, S.K. Batra, and M.P. Ponnusamy (abstract)
BMS-6 LAMA5-MEDIATED CAF-CANCER CELL CROSSTALK PROMOTES PANCREATIC CANCER AGGRESSIVENESS. Annant B. Kaur, Seema C. Parte, Palanisamy Nallasamy, Zahraa Wajih I Alsafwani, Surinder K. Batra, and Moorthy P. Ponnusamy (abstract)
BMS-7 RNA SPLICING AS AN ESSENTIAL DRIVER OF EPITHELIAL-TO-MESENCHYMAL TRANSITION. Eric Zadok Mpingirika, Chaitra Rao, Robert A. Svoboda, Siddesh Southekal, Heidi M.Vieira, Dianna H. Huisman, Deepan Chatterjee, Chittibabu Guda, Kurt W. Fisher, Olga A Anczuków , and Robert E. Lewis (abstract)
BMS-8 POST-TRANSCRIPTIONAL CONTROL OF SRSF9 PROMOTES THE EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN COLORECTAL CANCER CELLS. Priyanjali Mukherjee, Chaitra Rao, Robert A. Svoboda, Siddesh Southekal, Deepan Chatterjee, Heidi M. Vieira, Dianna H. Huisman, Chittibabu Guda, Kurt W. Fisher, Olga A. Anczuków, and Robert E. Lewis (abstract)
BMS-9 ABERRANT GLYCOSYLATION: DRIVERS OF PANCREATIC CANCER METABOLISM AND AGGRESSIVENESS. Wyatt Petersen, Frank Leon, Venkatesh Varadharaj, Zahraa Alsafwani, Surinder K. Batra, and Moorthy P. Ponnusamy (abstract)
BMS-10 UNIQUE GLYCOSYLATION PATTERN GOVERNS METASTATIC ORGANOTROPISM OF PANCREATIC DUCTAL ADENOCARCINOMA. Venkatesh Varadharaj, Frank Leon, Palanisamy Nallasamy, Wyatt D. Petersen, Surinder K. Batra, and Moorthy P. Ponnusamy (abstract)
BMS-11 HUMAN BLOOD CELL ISOLATION: THE CRITICAL FIRST STEP IN OUR LABORATORY’S IMMUNOBIOLOGY EXPERIMENTAL PROTOCOLS. Victor Rivero and Paul W. Denton (abstract)
BMS-12 DRUG PREFERENCE OF REMIFENTANIL, METHAMPHETAMINE, AND OPIOID/STIMULANT MIXTURE USING CHOICE PROCEDURE. Jakob Schmit, Mathew Meyer, Rachel Burrichter, Katie Shumacher, Kate Graham, Rachel Heueisen, Carissa Besonen, Chris Kang-Harris, Cece Nikodem, and Vanessa Minervini (abstract)
BMS-13 COMPUTATIONAL MODELING OF QUANTUM DOTS FOR NPRT USING MATLAB. Jackie Austin, and Dr. Andrew Ekpenyong (abstract)
BMS-14 AIR FORCE RELEVANT COLD INJURY INDUCES RELEASE OF MICROVESICLE PARTICLES WHICH IS DOWNREGULATED BY TREATMENT WITH TRICYCLIC ANTIDEPRESSANT IMIPRAMINE. Philip Richardsen, Christine Rapp, Karen Henkels, Jeffrey Travers, and Saber Hussain (abstract)
BMS-15 THE EFFECTS OF ON IGF-1 TENOCYTES UNDER TENDINOPATHIC ENVIRONMENTS - AN IN VITRO STUDY. Nicholas Lembke and Jeffrey Kiiskila (abstract)
BMS-16 VASCULARIZATION OF IN VITRO ORGANOTYPIC SKIN RAFT TO STUDY THE LYME DISEASE PATHOGEN. Jaxon Kramer, Trenten Theis, Carley Conover, Maddie Love, and Amanda Brinkworth (abstract)
BMS-17 THE TISSUE LEVEL OUTCOMES OF ALTERING THE NUCLEAR TO CYTOPLASM RATIO OF ENTEROCYTES IN INTESTINAL VILLUS. Rosemary Mwithiga and Subu Ramanathan (abstract)
STAPHYLOCOCCUS AUREUS PERSISTER CELLS EXHIBIT HIGHER TOLERANCE TO INNATE IMMUNE COMPONENTS
Kenan Brodd1, Emma Weis1, Alexis Hobbs1, Kimberly A. Carlson1, Austin Nuxoll1 broddk@lopers.unk.edu
1 - Department of Biology, University of Nebraska-Kearney, Kearney, NE.
Staphylococcus aureus is a pathogenic bacterium capable of causing serious infection in humans, like skin lesions, endocarditis, and sepsis. Difficulty treating infections may be due to the presence of persister cells. Persister cells are defined by surviving antibiotic treatment, however, it is unclear whether they have a fitness advantage to other stressors. Specifically, survival to innate immunity remains largely unexplored. Previous experiments show that a fumC (fumarase C, a tricarboxylic acid cycle gene) knockout exhibits increased survival to antimicrobial peptides. These experiments prompted further investigation of persister survival to other components of innate immunity such as reactive oxygen (ROS) and nitrogen (RNS) species. S. aureus strains of wild type HG003 and fumC::N∑ were grown to mid-exponential phase, challenged with paraquat (induces ROS) and NaNO2 (induces RNS), and survival was measured over 72 hours. Based on the finding that the fumC::N∑ strain had increased survival in the presence of ROS and RNS, survival within a macrophage was examined. RAW264.7 macrophages were infected with HG003 and fumC::N∑ and bacterial survival was measured over 48 hours. The fumC::N∑ strain exhibited increased survival suggesting persisters may provide a survival advantage to components of innate immunity in addition to antibiotics. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
NK CELL PHENOTYPING USING TWO COMPLEMENTARY FLOW CYTOMETRIC ANALYSIS STRATEGIES
Isabelle S. Weber1, Maia M.C. Bennett-Boehm1,2, and Paul W. Denton1, isabelleweber@unomaha.edu
1 - Department of Biology, University of Nebraska at Omaha, Omaha, NE;
2 – Department of Information Science and Technology, University of Nebraska at Omaha, Omaha, NE.
Natural killer (NK) cells are a component of the innate immune system, which functions to kill disease cells. NK cells achieve this in one of two ways: direct killing or antibody dependent cell-mediated cytotoxicity. With stimulation/activation, NK cells can experience changes in their surface phenotype due to changes in the composition/ratio of specific proteins (markers) present on the cell surface. These changes are related to the function of a cell. For example, cytokine-producing cells and regulatory cells normally have distinct surface phenotypes. Typically, we measure phenotype changes in terms of differences in the presence/absence and frequency of a specific marker(s). We have the capacity to measure the surface phenotype changes by using fluorescently labeled antibodies that specifically bind to a given marker. Then we use a flow cytometer to detect the fluorescence from the bound antibodies. Using our laboratory’s immunophenotyping panel, we have begun examining the dynamics of these immunophenotypes, including changes that occur in the presence of cancer cells used in the laboratory as target cells for NK cell-mediated killing. Typically, we analyze our flow cytometry data in a software platform called FlowJo. My project is. To couple our FlowJo analyses with a cloud-based analysis software called Cytobank to identify additional phenotypic characteristics that can only be found using such network analysis strategies. With this analysis strategy established, our goal is to apply this approach to improve our ability to relate the functional outcomes of human NK cell-mediated killing studies with surface phenotypes. Data obtained to date will be presented. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
SURFACE CD16 MODULATION BY TOLL-LIKE RECEPTOR 9 (TLR9) AGONISM ON HUMAN NATURAL KILLER CELLS
Angela N. Truong1, Anna R. Mahr, M.S.1, Maia M.C. Bennett-Boehm1,2, and Paul W. Denton, Ph.D.1, atruong@unomaha.edu
1- Department of Biology, University of Nebraska at Omaha, Omaha, NE;
2- Department of Interdisciplinary Informatics, University of Nebraska at Omaha, Omaha, NE.
Human CD16, a Fc-gamma-III receptor, binds to the constant fragment (Fc) of IgG (gamma) antibodies and leaves the Fab, or antigen-binding fragment, ends of the antibody free to bind to its specific antigen. An antibody’s structure is associated with a Y-shape. The two binding sites at the top of this “Y” are the Fab ends and the singular binding region at the bottom is the Fc end. When one or both the Fab binds to its specific target, the Fc is free to bind to an Fc receptor. This series of bindings is critical for a killing mechanism mediated by human natural killer (NK) cells called antibody-dependent cell-mediated cytotoxicity (ADCC). In NK cell-mediated ADCC, an IgG antibody’s Fab binds to a pathogenic cell’s marker while the Fc binds to a Fc receptor on a NK cell. In the Denton Immunobiology Laboratory, we observed an unexpected circumstance where an immunotherapy drug, a toll-like receptor 9 (TLR9) agonist, reduced CD16 surface levels on NK cells. Due to this reduction, the immunotherapy did not boost the NK cells’ ability to perform ADCC as suggested by other research groups in prior publications. In this talk, data will be presented regarding the impact of TLR9 agonism on CD16 expression and how this could be relevant for the use of TLR9 agonism in anti-cancer clinical contexts. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
DEVELOPMENT OF COMPUTATIONALLY DESIGNED, ADNOVIRAL-VECTORED INFLUENZA A VACCINES TARGETING NEURAMINIDASE
Amber Gadeken1, Lauren Sturgell1, Matt Pekarek1, and Dr. Eric Weaver1, agadeken2@huskers.unl.edu
1 - Department of Virology, University of Nebraska-Lincoln, Lincoln, NE.
Influenza viruses are responsible for yearly seasonal epidemics, causing acute respiratory illness and, in severe cases, multiple organ complications. To combat these seasonal epidemics, current vaccines target specific strains of influenza predicted to circulate in the upcoming year. However, these vaccines only induce strain-specific immune responses and provide limited cross-protection against mismatched strains. This limitation is more pronounced in influenza A strains (IAV), which exhibit broad genetic diversity, often leading to low efficacy of current vaccines. IAV also has the ability to cross-species jump into human populations, as seen in the 2009 swine flu pandemic. This project focused on designing a vaccine capable of eliciting immune responses to a broad range of human IAV and also cross protecting against cross-species transmission. Recent data has shown that targeting the influenza surface glycoprotein, neuraminidase, may provide protection against a wider variety of influenza strains and lead to a broader immune response in recipients. To accomplish this, our study focused on developing a centralized consensus N1 gene based on avian, swine, and human N1 sequences (CashN1) and a mosaic human N1 gene targeting T-cell epitopes. These computationally derived, neuraminidase targeting genes, are being cloned into a replication-defective human Adenovirus type 5 (rdAd5) genome via the pAdEasy Adenoviral Vector System. They will then be transfected into HEK 293 cells to produce replication-defective Ad5 viral particles, which will be used to produce viral vectored vaccine particles. In the future, these vaccines will be used for small animal preclinical trials and immune correlate studies. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
INVESTIGATION OF MAMMALIAN IFITM3 GENES AS VIRAL RESTRICTION FACTORS OF HSV-1 INFECTION.
Hayden Klaus1, Dane Bowder1, hayden.klaus@doane.edu
1 - Department of Biology , Doane University, Crete NE.
Herpes Simplex Virus type one (HSV-1) infections are some of the most common viral infections of humans in the world. HSV-1 is an enveloped virus which establishes latency in its host as a way to evade the host’s immune system. During viral infections host cells express viral restriction factors to halt infection to other cells. Cells express interferon in response to viruses and as a result increase expression of viral restriction factors, one such group of restriction factors is known as interferon-induced transmembrane (IFITM) family of proteins . Many stages of the viral replication cycle are inhibited by viral restriction factors; IFITM3 is thought to modulate cell membranes or antagonize viral structure and inhibit entry. IFITM3 specifically has been linked to potently restrict enveloped viruses like Human Immunodeficiency Virus and Influenza. Both human IFITM1 and IFTIM3 have been shown to strongly inhibit HSV-1 infection in vitro. Previous studies have shown that non-primate mammalian genes have significantly inhibited HIV and SIV infections which is the basis of our experiments on the inhibition of HSV-1. No research, to our knowledge, has been conducted into non-primate mammalian IFITM genes in the restriction of the HSV-1 virus. To investigate this, we intend to overexpress IFITM3 proteins in Vero cells followed by HSV-1 challenge and measure infection compared to controls. We anticipate that IFITM3 proteins from non-primate mammals will retain their restrictive ability during HSV-1 infection, due to protein and sequence similarity with the human IFITM3 ortholog.
KNOCKING OUT THE IMMUNITY REPRESSOR GENE IN THE PHAGE JABITH AND THE INTEGRASE GENE IN THE PHAGE LILHOMIEP HAS THE POTENTIAL TO CHANGE THE PHAGE LIFE CYCLE
Tiffany Carnahan1 and Erin Doyle1, tiffany.carnahan@doane.edu
1 - Department of Biology, University of Doane, Crete, NE.
Phage therapy is the use of bacteriophage to kill and target a specific bacteria. Antibiotic resistance of bacteria is an increasing problem, a solution is the use of bacteriophage for phage therapy. Bacteriophage has many advantages compared to antibiotics, including that bacteria will be less likely to become resistant to the phage. However, bacteriophages are host specific, which means that one phage is unable to kill all bacteria. Bacteriophages go through either a lytic cycle or lysogenic cycle. Phages that use the lytic cycle are more suitable for phage therapy because these phages immediately begin the process of phage replication, quickly leading to lysis of the host bacteria. Lysogenic phages are less useful in phage therapy because they may allow the host bacteria to survive and replicate after the initial phage infection. I am currently working with two lysogenic phages, Jabith and LilHomieP. Both phages were isolated using the host bacteria Mycobacterium smegmatis mc2155 from environmental samples collected at Doane University. I will use BRED, Bacteriophage Recombineering of Electroporated DNA, to knock out the immunity repressor gene from Jabith and the integrase gene from LilHomieP. My hypothesis is that by knocking out these genes that are associated with the lysogenic life cycle, will show more lytic infection characteristics. Once the mutant phages are isolated, we will measure plaque morphology, latent time and burst size to detect changes in the infection process. Successful genetic engineering of lysogenic phages will increase phages that have already been discovered to be used in phage therapy.
MORNING SESSION-B1
STRUCTURAL STUDIES OF A EUKARYOTIC OAZ1-PK RNA
Andree Kakish1, Lindsay Nedungadi1, Zach Frevert1, Lucas Struble2, Gloria Borgstahl2 and Juliane Soukup1
1 - Department of Chemistry, Creighton University, Omaha, NE;
2 - Eppley Cancer Institute, Nebraska Medical Center, Omaha, NE.
Riboswitches are elements found within noncoding regions of messenger RNAs (mRNAs) that regulate gene expression via metabolite binding. Upon binding to the riboswitch, the metabolite/ligand induces a conformational change in the RNA, resulting in modulation of the expression of a nearby gene. Most riboswitches are found in bacteria, although the Soukup lab has been investigating a highly conserved eukaryotic RNA sequence that exhibits the characteristics of a bacterial riboswitch – that being high specificity and affinity for a single ligand, the induction of RNA conformational change upon ligand binding, and a resulting change in gene expression that is ligand-dependent. The lab’s previous results demonstrate riboswitch function of a mouse RNA element that is highly conserved among vertebrate genes required for polyamine biosynthesis. The vast majority of biological organisms are able to synthesize natural polyamines (putrescine, spermidine, and spermine), which are essential for normal cell growth. Halting the production of polyamines prevents cell growth. Because of its unique regulatory function, this putative riboswitch RNA has potential biomedical applications, including anticancer pharmaceuticals. The main goal of my research project is to examine the structure and function of the Ornithine Decarboxylase Antizyme Pseudoknot (OAZ1-PK) RNA, a putative riboswitch, using X-ray crystallography. In doing so, my progress on the project includes large scale in vitro transcription of RNA, gel electrophoresis and size-exclusion chromatography (SEC) to purify the RNA, dynamic light scattering (DLS) to analyze the RNA and manual setup up of crystallography trays to test a wide array of chemical conditions for crystal growth.
OPTOELECTROCHEMICAL METHOD FOR READING DNA MICROARRAY ON AN AU MONOLITH ELECTRODE
Keegan Nitsch1, Ravi F. Saraf1, Akshat Saraf1, Jay Min Lim1, Joydip Dey1, Khirul Alam Titu1, knitsch3@huskers.unl.edu
1 - Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE.
Circulating miRNA is a promising biomarker for the early detection of cancer, cardiovascular, neurological, and behavioral diseases and for personalized medicine by determining therapy efficacy. The electrochemical beacon method can detect these miRNAs using a microarray of immobilized ssDNA probe with redox active dye attached at the free-end. The target miRNA’s are quantitatively measured by change in the redox signal of the dye. Specifically, probes that bind their complimentary target sequence from a liquid sample will hold the dye further from the electrode due to the rigidity of the double stranded molecule. causing a reduction in the redox signal. A novel differential reflectometer called Scanning Electrometer for Electrical Double-layer (SEED) will be described to measure local redox of the dye to quantify the probe-target binding. SEED allowed quantification of the target from 10 aM to 0.1 nM of target with 100% specificity vs. five non specific miRNAs. Conventional electrochemical beacon analysis has a Limit of Quantification in the picomolar range. The effect of buffer ions, specifically the chemisorption nature of the anions to Au electrode will be discussed to improve the performance of SEED. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
STRUCTURAL AND DYNAMIC STUDIES OF THE INTERACTION BETWEEN TWO PROTEINS INVOLVED IN GENE-SILENCING
Emily Ekstrum1, Evan Veltri1, Molly Dolan1, Zachary Frevert2, and Lynne Dieckman1, emilyekstrum@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE;
2 - Department of Biochemistry, University of Iowa, Iowa City, IA.
After DNA replication, newly synthesized DNA strands are wrapped around histone proteins to form nucleosomes during a process called replication-coupled nucleosome assembly. Two major proteins that function in replication-coupled nucleosome assembly are proliferating cellular nuclear antigen (PCNA) and chromatin assembly factor 1 (CAF-1). PCNA acts like a sliding clamp to encircle newly synthesized DNA and recruits CAF-1 to the replication fork. CAF-1 binds to PCNA and loads histone proteins onto the synthesized DNA to begin nucleosome assembly. This interaction between PCNA and CAF-1 is crucial for DNA packaging and gene regulation; however, the structural basis of this binding event is unknown. The goal of this study is to determine the structure and dynamics of the interaction between PCNA and CAF-1 using a combination of structural methods, including X-ray crystallography, small angle X-ray scattering (SAXS), and computational Langevin-based dynamics (LD) molecular simulations. We have carried out these structural studies with multiple PCNA-CAF-1 complexes, including a SUMO-modified form of PCNA. Thus far, we have generated microcrystals and P(r) curves from both SAXS and LD simulations of several PCNA-CAF-1 complexes. We are currently working to optimize the simulation-derived P(r) curves to better fit SAXS experimental P(r) curves. The comparison of the P(r) curves will validate SAXS data and support a structural ensemble determination between PCNA and CAF-1. Together, these studies will provide a comprehensive analysis of the structure and dynamics of the complexes formed between PCNA and CAF-1, and how these proteins regulate nucleosome assembly and gene silencing.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
SINGLE MOLECULE STUDIES OF PCNA AND CAF-1 USING TIRF MICROSCOPY
Olivia Nicholson1, Dr. Lynne Dieckman1, olivianicholson@creighton.edu
1 - Department of Biochemistry, Creighton University, Omaha, NE.
Shortly after replication, newly synthesized DNA is packaged and stored in structures called nucleosomes, which are the fundamental units of chromatin. Nucleosomes are composed of double-stranded DNA wrapped around eight histone proteins. This packaging process, called replication coupled nucleosome assembly, is crucial for protecting DNA and maintaining genomic stability. The level of compaction between these nucleosomes also determines which genes are expressed and which are silenced. Genomic regions of loosely packed nucleosomes are typically expressed, while regions of tightly packed nucleosomes are usually silenced. Two proteins that play major roles in replication coupled nucleosome assembly are chromatin assembly factor-1 (CAF-1), a heterotrimeric histone chaperone protein that deposits histone proteins onto DNA for nucleosome assembly, and proliferating cell nuclear antigen (PCNA), which binds and recruits CAF-1 to the replication fork. The interaction between PCNA and CAF-1 is essential for proper DNA packaging and gene silencing; however, the specific mechanism of binding between these two proteins is not known. We are using single molecule total internal reflection fluorescence (TIRF) microscopy to determine the binding kinetics and affinity of the interaction between CAF-1 and PCNA. We have built a TIRF microscope system, verified the attachment of proteins to the surface of a microscope slide, and optimized these assays to capture the interaction between individual PCNA and CAF-1 molecules. We are utilizing TIRF assays to determine the binding kinetics of the PCNA-CAF-1 interaction using varying concentrations of wildtype proteins. Future studies will use mutant forms of PCNA and CAF-1 to map their specific site(s) of interaction. These studies will increase understanding of the nucleosome assembly process and provide further insight on the importance of the PCNA-CAF-1 interaction in preserving genomic stability.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
EXAMINING THE EFFECTS OF TRADITIONAL CULTURING TECHNIQUES ON FILAMENTATION ASSAYS IN THE FUNGUS CANDIDA ALBICANS
Ethan Funke1 and Jill Blankenship1, efunke@unomaha.edu
1 - Department of Biology, University of Nebraska Omaha, Omaha, NE.
The fungus Candida albicans is both a commensal organism of the human microbiome as well as a major human pathogen. The ability of this fungus to switch between yeast-like and filamentous forms of growth is linked to pathogenesis. Our group has demonstrated that there are problems with the in vitro methods used to study this process. One of these issues, which our lab had not investigated in depth, was the standard use of culture tubes in liquid filamentation assays. Anecdotal evidence from our lab suggested that inducing filamentation in microscopy dishes yields higher rates of filamentation than the standard practice of growing cells in a culture tube and pipetting them onto a glass slide. This work will quantify discrepancies between the methods and the possibility that this discrepancy is due to a preferential selection of C. albicans in its yeast-like form when pipetting. We compare the use of each technique under ideal filamentation conditions, along with pipetting samples from both conditions to microscope slides, after different time points of incubation. Very preliminary data seems to support our hypothesis that pipetting skews the ratios of yeast:filamentous cells.
INVESTIGATING THE ROLE FOR DIACYLGLYCEROL IN HEAT TOLERANCE IN DROSOPHILA MELANOGASTER
Sunayn Cheku1, Blase Rokusek1, Sunanda Rajput1, Lawrence Harshman2, and Kimberly A. Carlson1, chekus@lopers.unk.edu
1- Department of Biology, University of Nebraska at Kearney, Kearney, NE;
2- School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE.
Since the discovery of the heat shock response, there is an interest in studying heat tolerance in Drosophila melanogaster. Thermal tolerance in Drosophila and other insects has become increasingly relevant given the expected increase in global temperatures due to climate change. The investigation of diacylglycerol (DAG) in heat tolerance is one of significant interest considering previous lipidomic studies within the context of thermal regulation. Evidence from a study by Ko and colleagues (2019) showed that lines selected for elevated temperature knockdown resistance had a statistically significant increase in different DAG levels. This exploratory project hypothesized flies and S2 cells administered DAG would show increased heat tolerance. Two different species of DAG, Disteoroyl glycerol (DSG) and Stearoyl arachidonoyl glycerol (SAG) were used. The experiments were conducted both in vivo and in vitro on adult D. melanogaster and S2 cells. Experimental groups included sex (female or male); temperature (39°C and 40.5°C), exposure time (6h, 12h, 24h, and 3/4d). Flies receiving either a DSG/PEG solid dispersion oral suspension compared to a PEG control demonstrated a trend for better heat tolerance in males at 40.5°C and in females at 39.5°C both at 24h post treatment, although not statistically significant (p > 0.06-0.1). Secondly, S2 cells were treated with 1mM DSG and SAG for 12 h and heat shocked at 39°C for 45 mins. Preliminary data suggests that cells treated with both the compounds show a significantly higher survival rate than untreated cells (p < 0.05) The significance of this exploratory study was the multiple parameters tested. If conclusive results are obtained, future research investigating the molecular mechanisms of DAG induced heat tolerance in Drosophila will be conducted. he project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #5P20GM103427.
MORNING SESSION-C1
THE ROLE OF SERUM ALBUMIN IN FILAMENTATION AND FARNESOL SEQUESTRATION.
Brigid M. Toomey1, Daniel J. Gutzmann1, Kenneth W. Nickerson1, and Audrey L. Atkin1 Btoomey2@huskers.unl.edu
1School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE
Candida albicans is an opportunistic pathogenic yeast that commonly colonizes the human gut and mucosal membranes. C. albicans exhibits polymorphism, and its morphogenesis between yeast and filamentous cells is associated with its virulence. A known inhibitor of filamentation is the hydrophobic molecule farnesol, which is produced by C. albicans throughout growth as a product of sterol synthesis pathways. A known stimulant of filamentation is blood serum, which contains the fatty-acid transport protein, albumin. The binding affinity of albumin for nonpolar, fatty-acid-like molecules suggests that it may interact directly with farnesol and influence morphogenic regulation. To further understand how albumin influences filamentation and farnesol localization, we quantified the activity and localization of farnesol in the presence of albumin. We found that farnesol accumulation and secretion increased in the presence of albumin. Additionally, we found that exogenous farnesol bioactivity in blocking germ tube formation was significantly decreased with added fatty acid-free albumin. Our findings suggest that albumin that is not bound to fatty acids has the capacity to bind to farnesol and sequester it from C. albicans, encouraging filamentation. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institute of Health under Grant #5P20GM103427.
INVESTIGATING THE POTENTIAL SURVIVAL ADVANTAGE OF STAPHYLOCOCCUS AUREUS PERSISTERS WITHIN A MACROPHAGE ENVIRONMENT
Carter Moss1, Emma Weis1, Kenan Brodd1, Alexis Hobbs1, Kimberly A. Carlson1, and Austin Nuxoll1, mossc2@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Staphylococcus aureus is a component of human microflora that typically exists benignly within the skin and nasal cavities of 30% of the population. However, under the right conditions, S. aureus acts as an effective opportunistic pathogen linked to inducing several diseases and nosocomial infections. Consequently, despite medicinal efforts, S. aureus has evolved various ways to circumvent both antibiotics and aspects of the innate immune system. Professional phagocytes, predominantly macrophages and neutrophils, are critical innate immune cells that interact with S. aureus, acting as a line of defense against the bacteria to resolve infection via phagocytosis. Yet interestingly in recent studies, S. aureus persisters, which are a subpopulation of cells with lower metabolic activity, have been shown to exhibit a survival advantage against innate immune components, namely antimicrobial peptides. Given this, we reasoned persisters may also possess a survival advantage to other components of innate immunity such as macrophages. To investigate this, we utilized a wild type S. aureus strain, HG003, and a high persister strain, fumC::NΣ to determine whether persisters were better able to survive within macrophages post phagocytosis. Furthermore, we employed a persister marker, Pcap5A::dsRed to monitor whether S. aureus cells with the highest expression of the persister marker are more adept to survival within a macrophage. Understanding S. aureus’s subversion and exploitation of these multifaceted innate immune system interactions may prove paramount for comprehending host-pathogen interactions. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
SEX DIFFERENCES IN INNATE IMMUNE RESPONSE FOLLOWING PEANUT EXPOSURE
Sunanda Rajput1 and Joseph Dolence1, rajput2@lopers.unk.edu
1- Department of Biology, University of Nebraska Kearney, Kearney, NE.
Knowledge of the immunological mechanisms involved in the development of peanut (PN) allergy remain unclear. Analysis of US adults showed that females were twice as likely to develop PN allergy during childhood, indicating sex hormones may regulate PN allergic responses. This study aims to understand the role of sex hormones in the response of mast cells (MCs) and Type 2 innate lymphoid cells (ILC2s) to PN. Previously, androgen receptor-deficient (ARTfm) mice showed higher PN-specific antibody and worse anaphylactic response to PN than WT males. WT males also displayed less severe anaphylactic response compared to ARTfm and WT females. We examined whether sex differences impacted the response of lung and peritoneal cavity MCs after 3-day inhalation exposure to understand how MCs initially respond to PN. Activated MCs have been shown to further activate ILC2s. Whether this occurs following PN exposure is unknown. After 3 days, we found no sex difference in lung and peritoneal MCs population in WT male, female, and ARTfm mice even though we showed ILC2s display a greater response to PN in female mice. To understand further, we used an 11-day PN inhalation model to investigate how sex differences impacted the response of lung ILC2s and MCs at a time point just before PN-specific adaptive responses begin to elicit PN-specific IgE. We found slight activation of the MC population in lung. Most striking however, was our finding that PN-activated ILC2s at 11 days displayed increased expression of TNFR2, suggesting MCs and ILC2 work together to drive PN-specific allergic responses. Recent studies have suggested that MCs work to further activate ILC2 through TNF-alpha. However, this connection has not been shown to drive immune responses to PN. Future studies will examine whether TNF-alpha produced by mast cells following PN exposure activates ILC2 in a TNFR2-dependent manner. Taken together, this data allows us to better understand the role of these cells in driving PN allergy. Such information is critical to develop novel therapeutic avenues for patients suffering from PN allergy.
EXPLORING MOMP AS A TARGET FOR AN mRNA CHLAMYDIA VACCINE
Parker Tinsley1, Shawn Pearcy1, Douglas Christensen1, patins01@wsc.edu
1- Department of Life Sciences, Wayne State College, Wayne, NE.
Chlamydia trachomatis is the most common STI reported in the United States and is an infection of high prevalence in many developing countries. This parasitic bacterium is not difficult to treat, but untreated women can spread C. trachomatis to newborn’s eyes causing trachoma. Major Outer Membrane Protein (MOMP), a surface protein expressed during both the elementary and reticulate body stages of Chlamydia infection, was identified as a strong vaccine candidate. Our lab has over-expressed MOMP in a prokaryotic host and purified the protein to approximately 95% purity. However, protein folding in reduced atmospheres of host prokaryotes may affect the proteins potential as a vaccine. Here we initiate the ligation independent cloning of the MOMP gene into a shuttle vector (pCDNA3-mRuby) to explore the possible transcription of MOMP mRNA in a eukaryotic host. If successful, eventual mRNA processing triggers will be incorporated to aid in evaluating this gene as a possible mRNA vaccine. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427
INVESTIGATING THE ROLE OF LINCRNA NOSTRILL IN ANTIVIRAL AND INFLAMMATORY RESPONSES IN NEURONS
Aaron Marta1, Sophie Ciechanowski2, Jodi Hallgren1, Hannah Pflum2, Paige Harty2, Davis Thulber2, Kirsten Drescher1, Annemarie Shibata2, aaronmarta@creighton.edu
1 - Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE;
2 - Department of Biology, Creighton University, Omaha, NE.
Viral infection in the brain can lead to significant neuronal damage and neurodegeneration. In the immune environment of the brain, neurons and microglial immune cells drive an antiviral response to effectively clear virus and promote the survival of neurons or ineffectively clear virus and promote neurodegeneration. Previous work in our lab has shown that viral infection of microglia with Theiler Murine’s Encephalomyelitis Virus (TMEV) upregulates an NFkB-dependent lincRNA Nostrill. Nostrill regulates Interferon Response Factor (Irf7) and Type 1 Interferon (Ifnα/β) gene transcription in microglia in vitro. In the TMEV mouse model system, neurons are infected before microglia. Depending on the strain of mouse infected with TMEV, acute infection is followed by viral clearance and neuronal survival or chronic viral infection and neuronal loss and demyelination. It is not known whether the antiviral response in neurons upregulates Nostrill and whether Nostrill is differentially expressed in strains of mice that develop acute or chronic TMEV infection. Therefore, I am investigating whether Nostrill is differentially expressed in neurons in acutely infected C57Bl6 mice and chronically FVB mice. Further I have developed an in vitro model system to evaluate the mechanism of Nostrill regulated gene transcription during neuronal antiviral immune responses. RT-qPCR evaluation of brain tissue isolated from TMEV-infected FVB mice at 7 days post infection demonstrated that Nostrill expression increased 0.35 ± 0.14fold (p=0.04, N=4, ±=SEM) compared to uninfected mice. Expression of inflammatory gene iNos increased 0.77 ± 0.35fold (p=0.04, N=4) and Ifnγ increased 9.51 ± 1.28fold (p<0.0001, N=4) compared to uninfected mice. In TMEV-injected FBV mice, Ifnα decreased 0.44 ± 0.15fold (p=0.01, N=4) and Ifnβ decreased 0.44 ± 0.15fold (p=0.01, N=4) compared to uninfected mice. Nostrill, iNos, Ifnγ, Ifnα, Ifnβ are not significantly increased in TMEV-infected C57 mice at 7 days post infection. In vitro studies utilize the human neuronal cell line SHSY5Y and mouse Neuro2a cell line. TMEV infection increased Nostrill and inflammatory cytokine expression in both cell lines in a time and dose dependent manner. SHSY5Y and Neuro2a cell lines will be used to examine the mechanism of Nostrill-regulated gene transcription in antiviral immune responses. Taken together, these data will determine whether the lincRNA Nostrill is important in regulating antiviral immunity and gene transcription and whether Nostrill is a potential target for modulating chronic inflammatory processes in neurons.
This publication was made possible by grants from the National Institute for AIDS and Infectious Disease (NIAID) (1 R15 AI156879) and the National Institute for General Medical Science (NIGMS) (5P20GM103427), components of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS or NIH.
INVOLVEMENT OF PUTATIVE MEMBRANE RAFT PROTEINS IN CAENORHABDITIS ELEGANS’ INNATE IMMUNE RESPONSE TO PATHOGENIC STENOTROPHOMONAS MALTOPHILIA
Minjae Song1 and Michael Herman1, msong6@huskers.unl.edu
1 - Herman Lab, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE.
Stenotrophomonas maltophilia is a gram-negative, multidrug-resistant bacterium capable of causing infections in immunocompromised patients. Caenorhabditis elegans is an excellent model organism in which to investigate the pathogenic mechanisms of S. maltophilia, as members of the Stenotrophomonas genus are naturally found in association with the gut microbiome. Genes significantly involved in C. elegans’ innate immune response to S. maltophilia were previously identified through RNA-sequencing analysis, three of which encode for membrane raft proteins irg-8, dod-19, and B0024.4. All three genes share paralogous sequence similarity, and irg-8 and dod-19 lie directly adjacent to each other in the C. elegans genome, suggesting that they arose from gene duplication. To investigate the implications of membrane rafts and duplicate genes in innate immune response, and to elucidate the mechanisms of S. maltophilia pathogenicity, these genes were chosen for further analysis. Current efforts focus on characterizing these genes further through a loss-of-function analysis and the generation of fluorescent fusion proteins. Loss-of-function analysis seeks to analyze the purpose of the possible redundant function of the three genes, which will be accomplished through the generation of double and triple knockout mutants. Fluorescent fusion proteins, on the other hand, will directly enable the observation of expression and localization activity, including subcellular localization. All strains will be generated using CRISPR-Cas9 genome editing technology. This presentation will report on a successfully generated fluorescent irg-8 strain demonstrating upregulated expression patterns in response to S. maltophilia. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
MORNING SESSION-D1
SCREENING OF PROMISING DRUG-LIKE COMPOUNDS FOR ACTIVITY AGAINST PARASITIC AMOEBA, NAEGLERIA
Brianna N. Davis1, Andrew J. Neville1, Jeff M. Zimmerly1, and Paul H. Davis1, briannadavis@unomaha.edu,
1 - Department of Biology, University of Nebraska-Omaha, Omaha, NE.
Naegleria fowleri is a pathogenic amoeba that causes primary amebic meningoencephalitis (PAM), a condition with a high mortality rate of 97%. While rare, the high mortality rate is due to the lack of FDA-approved drugs for treatment and ineffective current recommended treatments. While researchers are attempting to identify novel and repurposed drug-like compounds that could prove effective, the data are mainly limited to in vitro approaches. We propose that novel and repurposed drugs can be tested for efficacy in vitro and results confirmed and verified through in vivo approaches to push compounds closer to clinical development. Here, we review the findings of various potent and promising compounds in vitro and explore their further potential in additional in vivo studies.
ISOLATION OF CERE FROM WILLAERTIA MAGNA AND TETRAMITUS THORNTONI
Isaac Richards1, isrich01@wsc.edu
1 - Department of Biomedical Sciences, University of Creighton, Omaha, NE;
2 - Department of Biology, Wayne State College, Omaha, NE.
Primary amebic meningoencephalitis (PAM) is a rare and aggressive CNS disease with a 97% mortality rate. It is caused by Naegleria fowleri, known as the “brain-eating amoeba,” destroying brain tissue, typically leading to death within a few days of contraction. Many other species of closely related amoeba are non-pathogenic. One trait shared by the Naegleria genus, including N. fowleri, is the unusual location of rDNA on CERE plasmid in the nucleolus. By studying the rDNA of two species, Willaertia magna and Tetramitus thorntoni, we hope to provide the groundwork and supplement gaps in knowledge to build a more holistic understanding of these organisms for future studies. Currently one of the major aspects affecting the research is contaminating DNA from E. coli, which is used as a nutrient source for T. thorntoni. We are attempting different approaches to remove the E. coli genetic material at this time.
PEPTIDE AMPHIPHILES AS A NOVEL DRUG DELIVERY SYSTEM
Andrew Wegner1, Martin Conda-Sheridan1, Huihua Xing1, and Jiachen Feng1, ajw91438@creighton.edu
1 - Department of Pharmacy, University of Nebraska Medical Center, Omaha, NE.
Modern chemotherapeutic agents have an array of detrimental side effects, making treatment difficult for patients. These side effects occur because chemotherapeutic agents interact with healthy cells as well as cancerous ones. Ideally, drug delivery systems could be used to increase target specificity for chemotherapeutic drugs, minimizing harm to the patient while increasing the effectiveness of the treatment. One potential solution for such a delivery system may be peptide amphiphiles (PAs). PAs form biologically compatible nanostructures. The morphology of these nanostructures can be changed via the pH of the solution they reside in. Moreover, human physiological pH is 7.4 while the pH of the tumor microenvironment is 6.5. A PA nanostructure capable of storing a chemotherapeutic agent and undergoing a morphological transition from pH 7.4 to 6.5 could be developed, allowing a drug to be delivered to a tumor with high specificity. Currently, the relationship between solution pH and nanostructure morphology of systems containing multiple PAs is not well established. This study aimed to determine the effects of pH on PA nanostructure morphology for systems containing two different PAs. Two peptide amphiphiles were placed in solution, the pH was adjusted, and aggregates were allowed to form. The nanostructures of one system, C16K3/C16K2, were examined at pHs 7, 9, and 11 via transmission electron microscopy. The pH 7 sample formed only micelles. The pH 9 sample formed micelles but demonstrated a morphological transition to ribbons. Finally, the pH 11 sample formed only needle-like fibers. These results show that different PAs will interact with each other to form nanostructures. Additionally, when the pKa of one PA is reached (but not the other), the PAs begin to act more independently in solution. This information will further be considered to build a model capable of accurately predicting pH related morphological nanostructure changes.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
DIFFERENTIAL INTERACTIONS WITHIN THE GUT MICROBIOME OF MARMOSETS TREATED WITH ANTIBIOTICS AS REVEALED BY COMPUTATIONAL NETWORK ANALYSIS
Jordan B. Hernandez1,2,3, Shivdeep S. Hayer1,3, Sophie Alvarez4, Kathryn Cooper5, Mahsa Mohammadi7, Hyun-Seob Song1,6,7, and Jonathan B. Clayton1,3,6,8,9*, jordan.hernandez@unmc.edu
1 - Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE;
2 - Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE;
3 - Department of Biology, University of Nebraska at Omaha, Omaha, NE;
4 - Proteomics and Metabolomics Facility, Nebraska Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE;
5 - School of Interdisciplinary Informatics, College of Information Science and Technology, University of Nebraska at Omaha, Omaha, NE;
6 - Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE;
7 - Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE;
8 - Callitrichid Research Center, University of Nebraska at Omaha, Omaha, NE;
9 - Primate Microbiome Project, University of Nebraska-Lincoln, Lincoln, NE.
Antibiotics are an important tool in modern medicine that can be used to control the otherwise unmitigated growth of pathogenic bacteria, but their administration can have unintended consequences within the gut microbiome by pushing resident gut bacteria into an unhealthy state known as dysbiosis. While gut dysbiosis following antibiotic treatment is a well-documented phenomenon, the specifics of how the gut microbiota are affected – and how the microbiota affect their host in turn – is poorly understood. In this study we sought to answer these questions by using 16S sequencing to measure how bacterial abundance changes in marmoset monkeys treated with a broad-spectrum antibiotic cocktail for 28 days, followed by a 14-day recovery period. Results from k-means clustering performed on 16S data revealed the existence of 2 distinct subgroups within the antibiotic-treated marmosets: an antibiotic medium-resistance group that was less affected by antibiotics overall, and a low-resistance group that was more affected. Network analysis comparing the subgroups to each other and to vehicle-treated controls further revealed that the bacterial genus Acinetobacter had a high number of interactions (degree centrality) with other genera in antibiotic-treated marmosets, while the genus Roseburia had high degree centrality in control marmosets, suggesting that these genera play an influential role within the microbiomes of their respective treatment groups. Moreover, the genus Faecalibacterium had high degree centrality in the antibiotic medium-resistance subgroup, suggesting that this genus contributed to microbiome stability under antibiotic stress. Although differentially abundant metabolites were detected in both subgroups, the majority of those (72/107) were exclusively altered in low-resistance marmosets, indicating that more pronounced antibiotic-induced gut dysbiosis is accompanied by a corresponding change in the fecal metabolome. More specifically, impaired phenylalanine metabolism and an overabundance of sugar derivatives were observed in the low-resistance subgroup during antibiotic treatment. Together, these results suggest that reduced numbers of stability-promoting gut bacteria as a result of antibiotic treatment can cause dysregulation of microbially-mediated pathways responsible for carbohydrate fermentation and the synthesis of potentially neuroactive phenylalanine derivatives.
COMBATING FOSFOMYCIN RESISTANCE VIA INHIBITION OF FOSB WITH GLCNAC-MAL
David Johnson1 and Mary E. Keithly1, david.johnson@eagles.csc.edu
1 – Department of Physical Science, Chadron State College, Chadron, NE.
An understanding of the inhibitive power of GlcNAc-mal on FosB could provide insight into combating fosfomycin resistance by Gram positive organisms such as methicillin-resistant staphylococcus aureus (MRSA). MRSA infections are a growing global concern. MRSA infections in healthy adults are often asymptomatic. This leads to unchecked colonization of the bacteria among unsuspecting healthy people. MRSA’s principal victims are the immunocompromised and otherwise infirm. The unfortunate combination of MRSA’s widespread unnoticed colonization and its often-deadly infection of immunocompromised patients has led to MRSA becoming one of the most serious and feared hospital-acquired infections. MRSA is difficult to combat due to its intrinsic resistance to antibiotics. Enter Fosfomycin: by no means a novel antibiotic but a promising one in the fight against MRSA. Studies in vitro have shown that Fosfomycin is able to disarm MRSA’s destructive potential. However, in vivo Fosfomycin can be blocked from acting upon MRSA by the manganese dependent bacillithiol transferase enzyme FosB. It is crucial, therefore, to find a way to inhibit FosB to allow Fosfomycin to deactivate MRSA. GlcNAc-mal is a precursor in the biosynthesis pathway for bacillithiol, the FosB substrate. GlcNAc-mal lacks the thiol group necessary for FosB activity and may serve as a competitive inhibitor of FosB. Kinetic analysis of in vitro FosB assays in the presence and absence of GlcNAc-mal are in process. If inhibition is successful, this is the first step toward novel drug therapies that could be used to defeat MRSA.
EFFICIENCY OF TOOTH WHITENING PRODUCTS AND THE EFFECT ON THE TOOTH
Ashley Christianson1, Mary Keithly1, ashley.christianson@eagles.csc.edu
1- Department of Physical Sciences, Chadron State College, Chadron NE.
Whitening teeth has become a constant consumer demand and continues to become popular in society. As whitening products become more popular it is important to determine if the tooth structure is altered or damaged, along with comparing the efficacy of whitening products for consumers. Coffee is also widely consumed, despite the possible health effects. Coffee intake can vary, but over 100 million U.S. adults drink coffee. In this study, the effect of whitening properties and tooth structure will be observed on extracted human teeth after a staining process using coffee. Before the staining process takes place, pictures were taken along with the mass of each tooth. A pot of coffee was made using 20g of coffee grounds and 500mL of water. The teeth were placed into a plastic cup containing enough coffee to cover the entire tooth. The teeth sat in coffee for two hours each day. They were brushed with the assigned toothpaste for 30 seconds each. Once complete the teeth were rinsed off and placed in artificial saliva. Every two weeks new pictures were taken along with recoding the mass of each tooth. Analysis of final images and masses is in process and full results will be presented.
MORNING SESSION-A2
ANALYZING KILLING FUNCTIONS OF HUMAN NATURAL KILLER CELLS THROUGH A TRANSCRIPTIONAL LENS VIA SINGLE-CELL RNA SEQUENCING
Cami R. Bisson1 and Paul W. Denton1, camibisson@unomaha.edu,
1 - Department of Biology, University of Nebraska at Omaha, Omaha, NE.
Natural killer (NK) cells have a distinct role in the human immune system: to kill infected or cancerous cells. Two methods of killing are utilized to achieve this. The first, direct killing, occurs between an NK cell and its target cell via direct methods, where no antibody is needed for target cell recognition. The second, antibody-dependent cell-mediated cytotoxicity (ADCC), is facilitated by the presence of an antibody specific to the target cell. To assess killing capacities of human NK cells, we used an immunotherapy treatment, a toll-like receptor 9 agonist, to initiate an immune response. We then observed NK cell-mediated killing of our target cancer cell lines, K562 (direct) and Daudi (ADCC). Our experimental approach is a novel assay where we assess both methods of NK cell killing. Our data show that, with stimulation, direct killing increased while ADCC did not. The objective of the current experiment is to analyze the killing functions of natural killer cells through a transcriptional lens via single-cell RNA sequencing. The expression of important markers, such as CD69, a marker for NK cell activation, and other markers for cytotoxic potential, were analyzed in one representative donor sample. Our results follow changes in transcriptional levels within NK cells over four time points (0 to 60 hours) and may open doors into other facets of NK cell killing, both ADCC and direct, and how they correlate to recent findings. This preliminary data may also contribute to clinical trials utilizing immunotherapy treatments involving NK cell-killing abilities.
SPANNING PARADIGMS: USING IMMUNOTHERAPY TO ENHANCE HUMAN NATURAL KILLER CELL ANTIBODY DEPENDENT CELL-MEDIATED CYTOTOXICITY AGAINST CANCER AND INFECTIOUS DISEASE.
Jaden Nienhueser1, and Paul W. Denton PhD1
1- Department of Biology, University of Nebraska at Omaha, Omaha, NE.
A primary goal of the Denton Immunobiology laboratory is to evaluate immunotherapy strategies with the goal of improving the killing capacity of human natural killer (NK) cells. To date, this work has been in the context of killing cancer cells. Malignancy is a paradigm of disease that has a nearly immeasurable scope. However, another disease paradigm has a similarly large sphere – infectious diseases. The goal of this project is to determine whether immunotherapy findings in a cancer context can span paradigms and similarly impact treatment approaches in an infectious disease context. Our goal is to perform infectious disease-related experiments without the need to incorporate fully infectious agents into our experimental approach. To do this, we obtained cells that express human immunodeficiency virus (HIV) envelope proteins constitutively. The reason that we chose HIV as the pathogen to represent the infectious disease paradigm in our studies is the fact that HIV treatments exist but are not curative. Our goal is to contribute to many research efforts focused on helping to “train” the immune system to fight HIV in the absence of other treatments (e.g., antiretroviral therapy). The target cells with HIV protein on their surface appear to be infected from the perspective of the human NK cells that we have treated with immunotherapies. To allow the NK cells to recognize the presence of HIV protein, we use an antibody that is specific for the envelope and capable of directing the NK cell to perform the killing function known as antibody-dependent cell-mediated cytotoxicity (ADCC). Data to date will be presented. The project described was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
DEVELOPMENT OF A NOVEL ASSAY TO ASSESS MACROPHAGE PHENOTYPE
Furqan Mahdi1, Kayla Ney1, and Rebecca Wachs1, fmahdi2@unl.edu
1 - Department of Biological Systems Engineering, University of Nebraska Lincoln, Lincoln, NE.
The role of macrophages in inducing inflammation within the spine due to disc degeneration has garnered significant attention in orthopedic research. Inflammation from macrophages can lead to the development of chronic low back pain, which creates a low-quality lifestyle. A pivotal study area is understanding the polarization of macrophages, which determines their pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. Currently, immunocytochemistry (ICC) is the gold standard for quantifying the polarization of macrophages. When a macrophage adopts the M1 phenotype, it produces nitric oxide synthase (iNOS), which, in turn, catalyzes the digestion of arginine into nitrite. Additionally, M2 macrophages produce arginase, an enzyme that converts arginine into urea. The ICC method stains for iNOS and arginase, but it is a time-intensive process, spanning two days, and a costly method. Furthermore, ICC is an endpoint assay requiring cell fixation, limiting further investigation of life cells. We hypothesized that we could effectively assess macrophage polarization using commercialized kits to measure urea and nitrite production in cell media post-cell culture. As mentioned, urea and nitrite are the endpoints of the metabolic pathways of arginine digestion. Female rat primary bone marrow macrophage cells were cultured and differentiated into M1 and M2 phenotypes using IFN-gamma, LPS, IL-4, and IL-10 cytokines. We compared commercialized urea and nitrite kits to the standard ICC method through the following steps: (1) preparing urea and nitrite standards in a 96-well plate, (2) adding reaction buffers, (3) incubating, and (4) recording measurements with a plate reader. Simultaneously, we performed ICC staining on cells. This approach allows for a direct comparison between the kits and ICC. Our investigation revealed a strong correlation between the commercialized nitrite kit and ICC staining, displaying elevated nitrite levels in M1 macrophage samples, in line with increased iNOS presence. Likewise, the urea kit results are aligned with ICC staining, indicating elevated urea levels in M2 macrophage samples and high arginase expression. These results affirm the reliability of these kits for quantifying macrophage polarization through nitrite and urea measurements, presenting them as viable ICC alternatives. In conclusion, our study validated the feasibility of using commercialized kits for rapid, cost-effective, and dependable macrophage polarization assessment. These findings can advance chronic low back pain research and help discover a treatment for back pain caused by inflammation.
SNIFFING OUT PROINFLAMMATORY FACTORS – MODULATION OF MICROGLIAL INFLAMMATORY RESPONSE THROUGH NOVEL LONG NON-CODING RNA
Sophie Ciechanowski1, Aaron Marta3, Olivia Burleigh1, Nicholas Mathy2, Kristen Drescher3, and Annemarie Shibata1, sophieciechanowski@creighton.edu,
1 - Department of Biology, Creighton University, Omaha, NE;
2 - Creighton University, School of Medicine, Omaha, NE;
3 - Creighton University, Department of Medical Microbiology and Immunology, Omaha, NE.
Neurodegenerative pathologies of various diseases are associated with systemic immune responses triggered by viral and bacterial infections. When activated by pathogens, microglia participate in pathogen clearance and promote neurorecovery or cause neurotoxicity. An increased understanding of microglial antiviral mechanisms is important to identify potential targets for limiting neurotoxicity and neurodegeneration. Our previous work shows that long noncoding RNAs (lncRNAs) can regulate microglial antibacterial responses. We hypothesize that lncRNAs play an essential role in regulating microglial antiviral immunity. We used in vivo, ex vivo, and in vitro mouse model systems to show that infection with Theiler’s murine encephalomyelitis virus (TMEV) upregulates expression of the lncRNA Nostrill in chronically demyelinated brain (~4fold), infected CNS microglia (~3fold), and microglial cell lines (~3fold), as compared to controls (n=3, p<0.05). Silencing of Nostrill in microglial cell lines increases viral burden and reduces the expression of the antiviral genes Irf7 and Ifnβ. Overexpression of Nostrill reduces viral burden and increases antiviral genes Irf7 and Ifnβ. LncRNA Nostrill may influence microglial antiviral immunity by regulating their polarization and antiviral activation state. IL-6, CD45, and IL-10 are genes expressed in microglial antiviral activation states and are all upregulated in microglia following TMEV infection (~2fold). Silencing Nostrill blocks the upregulation of these activation markers. Due to the role of microglia in CNS immunity, studies investigating the mechanisms by which microglia regulate their polarization and immune activity are essential in the effort to identify potential targets for limiting microglial-associated neuronal cell death in CNS proinflammatory disease states.
This publication was made possible by grants from the National Institute for AIDS and Infectious Disease (NIAID) (1 R15 AI156879) and the National Institute for General Medical Science (NIGMS) (5P20GM103427), components of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS or NIH.
NOSTRILL MEDIATED BEHAVIORAL DIFFERENCES FOLLOWING ACUTE AND CHRONIC INDUCED DEMYELINATING DISEASE PATHOLOGIES
Hannah Pflum1, Jodi Hallgren2, Sophie Ciechanowski1, Aaron Marta2, Paige Harty1, Kristen Drescher2, Annemarie Shibata1, hmp50129@creighton.edu
1 - Department of Biology, Creighton University, Omaha, NE;
2 - Creighton University, School of Medicine, Omaha, NE.
Systemic inflammation following viral infection can result in the development of neurodegenerative pathologies, as seen in disorders such as multiple sclerosis (MS). Microglia play an essential role in the innate immune response in the CNS, and microglial activation following pathogenic exposure can result in both neurorecovery or neurotoxicity. Long non-coding RNAs (lncRNAs) are transcribed RNAs that regulate processes via interaction with RNA-binding proteins, such as transcription factors, instead of coding for proteins. LncRNA upregulation has been observed in neurodegenerative disease states, such as within the lesions and blood serum of MS patients. We hypothesize that the novel lncRNA Nostrill plays a role in regulating microglial antiviral immunity in vivo and is differently expressed in mouse strains with variable abilities to clear viral pathogens following infection. Theiler’s murine encephalomyelitis virus (TMEV) is a single stranded RNA cardiovirus that induces infectious demyelinating disease (IDD) pathology in mice that is similar to MS in humans. We used the TMEV-IDD in vivo mouse model system to monitor the behavior of acute (C57BL/6J) and chronic (FVB/nJ) neurodegenerative MS-like disease. Male and female C57BL/6J and FVB/NJ mice were intracranially injected with either 10pfu of TMEV in 10μl HBSS or 10μl of HBSS alone. Behavioral testing began at 7-day, 35-day, and 90-day postinjection. Recorded videos were used for quantification and behaviors were analyzed by three blinded reviewers. After behavioral studies, the mice were euthanized and brain, spleen, and spinal cord tissue was collected for RT-qPCR and in situ analyses. Infected C57BL/6J female mice maintain relatively steady levels of mobility as determined by the cylinder test when comparing data across the time points. Infected FVB/nJ mice show a decrease in mobility between the 35-day and 90-day time point. Interestingly, RT-qPCR analyses of C57BL/6J and FVB/nJ mice show that the lncRNA Nostrill is significantly upregulated in the cortical tissue of FVB/nJ mice at 7 days postinjection (n=6, 0.2810±0.1070, p<0.05) and 35-day point shows an increase in Nostrill expression in the spinal cord (n=6, 0.6746±0.1686, p<0.05). The FVB/nJ mice at the 35-day point show an increase in Nostrill. These data provide preliminary data suggesting a correlation between Nostrill upregulation and chronic neurodegenerative disease state involving microglial activation. Further studies are needed to understand the underlying molecular mechanisms of virally induced neuronal damage seen in the TMEV-IDD model system and neurodegenerative diseases.
This publication was made possible by grants from the National Institute for AIDS and Infectious Disease (NIAID) (1 R15 AI156879) and the National Institute for General Medical Science (NIGMS) (5P20GM103427), components of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS or NIH.
MORNING SESSION-B2
A NOVEL INTERACTION BETWEEN TWO PROTEINS INVOLVED IN NUCLEOSOME ASSEMBLY
Grace Jaworski1 and Lynne Dieckman1, gracejaworski@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE.
Immediately following DNA replication, DNA condenses around histone proteins into structural units called nucleosomes. The level of compaction of nucleosomes dictates whether genes encoded in the DNA are expressed or silenced. The process of packaging DNA immediately following replication is called replication-coupled nucleosome assembly. This process requires two major proteins: proliferating cell nuclear antigen (PCNA) and chromatin assembly factor 1 (CAF-1). PCNA is the eukaryotic sliding clamp protein that binds to and surrounds DNA during replication. It plays a critical role in almost all aspects of DNA metabolism by recruiting and coordinating the activity of several other proteins also involved in these processes. CAF-1 is a histone chaperone protein that is recruited to the replication fork by PCNA and functions by depositing histones onto newly synthesized DNA at silenced regions of the genome. Although the interaction between CAF-1 and PCNA is critical for the proper silencing of genes, it is unknown how these two proteins interact to allow replication-coupled nucleosome assembly to occur. The goal of my work is to characterize the thermodynamics of the interaction between CAF-1 and PCNA using isothermal titration calorimetry (ITC). Thus far, I have measured the thermodynamics of a known interaction between PCNA and CAF-1. I have also identified a novel, secondary interaction region on each protein using a qualitative protein-protein binding assay. I am currently working toward determining the thermodynamics of this novel, secondary interaction region. Understanding how PCNA and CAF-1 interact at the thermodynamic level will provide insight into how the recruitment of CAF-1 to the replication fork by PCNA mediates nucleosome assembly, and therefore gene silencing.
CHARACTERIZATION OF THE NUCLEAR LOCALIZATION SIGNAL (NLS) OF ORF1 OF NORA VIRUS
Belle Turk1, Amanda J. Macke2, Darby J. Carlson1, Alexis M. Hobbs1, and Kimberly A. Carlson1 turkb@lopers.unk.edu
1Department of Biology, University of Nebraska at Kearney, 2401 11th Ave, Kearney, NE 68849
2Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198
Nora virus is a picorna-like virus that is endemic in Drosophila melanogaster and referred to as D. melanogaster Norovirus (DmNV). The genome of DmNV contains four open reading frames (ORFs) known as ORF1, ORF2, ORF3, and ORF4. ORF1, the focus in this study, has a role in RNA interference, RNAi, suppression through inhibition of the RNA induced silencing complex, RISC. This allows Nora virus to remain persistent in its host. Sequence analysis of ORF1 shows not one, but four potential putative bipartite nuclear localization signal (NLS) sites. The NLS 2 and 3 sites overlap and are considered together as one NLS or NLS 2. Knockout mutations for NLS 1, 2, 3, 1 & 2, 1 & 3, 2 & 3, and all three knockout mutations together (1, 2, & 3), were created and cloned into the pCR-TOPO vector. The mutants will be subjected to sequencing to verify that the intended mutations were created. The verified mutants will be subcloned into pEGFP, transfected into S2 cells, the nucleus stained with DAPI, and visualized using confocal microscopy. This study will let us determine the identity of the ORF1 NLS responsible for translocation of DmNV to the nucleus. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #5P20GM103427.
SHAPE ANALYSIS OF CRASSOTREA GIGAS OAZ-PK RNA.
Elizabeth Alberts1, Juliane Strauss-Soukup-Coauthor1,2, eca66743@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE
2 - Creighton University School of Medicine, Department of Biomedical Sciences, Creighton University, Omaha, NE.
A riboswitch is a piece of non-coding RNA that functions in downstream gene expression when bound to a metabolite. When a riboswitch interacts with its metabolite, they will undergo a conformational change that will affect the expression of genes downstream to its binding site. The outcome of this pathway is a change in the production of the same metabolite it binds to. The Soukup lab is researching the potential eukaryotic riboswitch in the Ornithine Decarboxylase Antizyme pseudoknot (OAZ-PK) RNA segment. Known riboswitches in bacteria have a significant effect on various metabolic pathways, providing a way to develop new antibiotic treatments. Identification of a similar non-coding RNA in eukaryotic species may provide a possible way to develop novel anti biological agents. My project focuses on studying a potential riboswitch in Crassostrea Gigas, a species of oyster. Specifically, Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) experiments are being used to analyze structural changes of this non-coding RNA segment when it interacts with various natural and non-natural polyamines. Analyzing these structural changes will aid in identifying this RNA segment as a riboswitch that could open the possibility of developing novel anti biological agents.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
CHARACTERIZING HIGH PERSISTER PHENOTYPES IN STAPHYLOCOCCUS EPIDERMIDIS CLINICAL ISOLATES
Mariam Garcia1, Kaitlyn Pineda1, Kael Kingery1, Alexis Hobbs1, Kimberly A. Carlson1, and Austin Nuxoll1, garciaescobarm@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Staphylococcus epidermidis is an opportunistic pathogen that typically resides within our normal skin flora and is primarily associated with causing disease in immunocompromised individuals. Often these infections are biofilm-mediated and associated with indwelling medical devices. Antibiotic treatment of these infections is often unsuccessful, leading to poor patient prognosis. One possible explanation for these observations is the presence of persister cells (a subpopulation of dormant cells). High persister isolates have been observed in other microbial pathogens such as Pseudomonas aeruginosa and Candida albicans. Recent work in the related pathogen, S. aureus, demonstrates that persister formation is dependent on energy depletion through the tricarboxylic acid (TCA) cycle. We hypothesized that high persister isolates occur in S. epidermidis clinical isolates through an energy-dependent mechanism. To further explore this, Drosophila melanogaster, the common fruit fly, was utilized to monitor survival and observe whether high persister cells have increased tolerance to components of the innate immune system. To observe the possibility of a correlation between high persister formation and a dysfunctional TCA cycle, extracellular acetate (as an indicator of TCA cycle activity) was measured in high and low persister isolates. Of the 17 isolates screened, seven correlated with high extracellular acetate concentrations and exhibited high antibiotic tolerance, and four of them exhibited low antibiotic tolerance. Preliminary data has demonstrated the correlational relationship between a dysfunctional TCA cycle and increased persister formation. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
USING 3D PRINTED DEVICES TO ELUTE AND CONCENTRATE S. CEREVISIAE DNA
Esmeralda Mendez1, Thi Huynh1, and Kristy Kounovsky-Shafer,1, mendezortizey@lopers.unk.edu
1 - Department of Chemistry, University of Nebraska-Kearney, Kearney, NE.
Identifying large variations in a genome can be cumbersome. However, using large DNA molecules that span the genomic variations aids in assembling. However, due to the DNA molecule’s large size, routine molecular biology techniques can break DNA. Therefore, a method is required to prevent the breakage of DNA during cell lysis and be able to concentrate DNA. S. cerevisiae was tested to determine how much DNA could be eluted and concentrated in the 3D-printed device. The DNA was stained with YOYO-1 dye to track the progression of DNA through the device. The 3D-printed device was affixed to a glass slide, and an acrylamide “roadblock” was used to slow down the progression of DNA. The DNA insert was added to the device, and DNA was eluted into the solution and concentrated in front of the acrylamide roadblock. The DNA sample concentration was measured to determine how much DNA was eluted.
MORNING SESSION-C2
STRUCTURAL ANALYSIS OF OAZ RNA IN NEUROSPORA CRASSA
Caitlin Sousley1, Emma Curran1, and Juliane Strauss-Soukup1, ecjs92554@creighton.edu
1 - Department of Chemistry, Creighton University, Omaha, NE.
Riboswitches are segments of non-coding RNA that bind cellular metabolites to alter expression of a downstream gene. This is accomplished by the riboswitch interacting with a specific ligand which induces a structural change that can then alter transcription, translation, or RNA processing of the downstream gene responsible for producing more of the metabolite. Riboswitch regulatory behavior in bacteria has been largely documented. However, eukaryotic riboswitch behavior pertaining to biosynthesis of polyamines remains uninvestigated. Polyamines are organic molecules that interact with DNA, RNA, and proteins to influence cell growth, proliferation, and DNA stability. Therefore, understanding riboswitch structure and riboswitch-ligand specificity will greatly improve the targeting and reprogramming of polyamine biosynthesis using non-natural compounds and synthetic riboswitches for medicinal and biotechnological applications. The potential eukaryotic riboswitch (OAZ RNA) is highly conserved across a variety of organisms. Previous work in the Soukup lab has found strong evidence for the presence of a riboswitch in the mouse OAZ RNA by studying riboswitch-ligand conformational changes. The goal of this work is to investigate the OAZ RNA from Neurospora crassa. In-line probing (ILP) is being used to characterize conformational changes in the OAZ RNA from N. crassa. Specifically, ILP is used to identify changes in the secondary structure of the RNA in the presence of varying concentrations of polyamine ligands, such as spermine and spermidine. By using varying concentrations of the polyamine and different types of polyamines, it is possible to determine whether the OAZ RNA demonstrates one of the key characteristics of a riboswitch, conformational changes upon ligand binding. Further experiments will aid in examining binding affinity and ligand specificity for this potential riboswitch.
MUCIN 5AC MODULATES CANCER-ASSOCIATED FIBROBLAST HETEROGENEITY THROUGH EPIGENETIC REGULATION
Rachel Kehrberg1, Xiaoqi Li1, Surinder K. Batra1, and Sushil Kumar1, Rachel.kehrberg@unmc.edu
1- Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
Pancreatic cancer (PC) is a deadly malignancy characterized by the expression of mucins and a highly desmoplastic stroma comprising up to 90% of the tumor. Cancer-associated fibroblasts (CAFs), a significant component of this stroma, contribute to cancer pathobiology through the secretion of extracellular matrix (ECM), growth factors, metabolites, and cytokines. It is now well known that CAFs are a heterogeneous population with both tumor-promoting and tumor-restraining roles; however, the source of this heterogeneity remains poorly understood. In PC, CAFs are derived from local pancreatic stellate cells (PSCs) and distant adipose mesenchymal stem cells (AD-MSCs). Emerging evidence suggests CAF maturation is mediated through epigenetic mechanisms, including DNA methylation and histone acetylation. Our previous study using murine models demonstrated the critical role of mucin 5ac (Muc5ac) in developing a heterogeneous stroma. However, the molecular mechanisms involved in AD-MSC and PSC maturation into different subsets of CAFs remain elusive. Here, co-culture with Muc5ac expressing, but not Muc5ac knockout, cancer cells or treatment with conditioned media from these cells increased the expression of CAF subtype markers, including inflammatory CAF (iCAF) markers (Il6, Cxcl1, and Cxcl2) and antigen-presenting CAF (apCAF) markers (Cd74 and H2-ab1) in primary AD-MSCs. Muc5ac carries tumor cytokines trapped in its glycan tree, contributing to the micro enrichment of C-X-C motif chemokine receptor 2 (CXCR2)-ligands at the cell surface. Pharmacological inhibition of CXCR2 decreased levels of CAF markers, suggesting that Muc5ac-trapped ligands induce AD-MSC CAF maturation via the CXCR2 signaling axis. Treatment of AD-MSCs and PSCs with conditioned media derived from Muc5ac proficient, but not deficient, cancer cells induced STAT-3 phosphorylation. Mechanistic study indicated that AD-MSCs epigenetically mature into CAFs by Muc5ac mediated upregulation of DNA methyltransferase 3b (DNMT3b) and ten-eleven translocation 1 (TET1) dioxygenase. Treatment of PSCs with conditioned media derived from Muc5ac proficient, but not deficient, PC cells increased histone H3K27 acetylation marks, a common histone mark of active transcription, which increases during CAF maturation. Similarly, higher immunoreactivity to H3K27ac was seen in LSL-KrasG12D, Pdx1-Cre (KC) pancreatic tissues compared to LSL-KrasG12D, Pdx1-Cre, Muc5ac-/- (KCM) pancreatic tissues, both in the ductal and stromal compartments. This study suggests the importance of Muc5ac in regulating CAF heterogeneity by modulating the biology of CAF precursor cells through epigenetic regulation. Further, it indicates that AD-MSCs and PSCs have distinct mechanisms of CAF maturation and unique CAF marker expression, demonstrating CAF heterogeneity based on cellular origins.
HISTOLOGICAL VALIDATION OF NADH FLIM ANALYSIS TO NON-INVASIVELY DETECT SKIN CANCER
Jonathan Li1, Alex Chen1, Kennedy A. Haase1, Jackson M. Laurent1, Maimuna Olow Nagey1, Derek A. Remitar III1, Abraham J. Saks1, Hannah Schloman1, Jinann A. Shoshara1, Zachary J. Smith1, Fiona Sun1, Jacob A. Sweet1, Jake S. Wakahiro1, Michael Nichols1, Laura Hansen2, jli67020@creighton.edu
1 - Department of Physics, Creighton University, Omaha, NE;
2 - Department of Biomedical Sciences, Creighton, Omaha, NE.
Squamous cell carcinoma is a form of nonmelanoma skin cancer that can be caused by damage to DNA and DNA repair mechanisms from UV light. Cancer cells are reported to preferentially perform glycolytic metabolism over oxidative. Our lab focuses on developing a method to detect skin cancer non-invasively using optical biomarkers within cells using a technique called fluorescence lifetime imaging microscopy (FLIM), which involves exciting molecules to higher energy states and measuring fluorescence decay time as the molecule returns to the original energy state. This technique can be used to visualize NADH, a biomarker crucial to cellular metabolism that exhibits differences in fluorescence lifetime depending on the metabolic activity done in the cell. NADH bound to proteins in oxidative metabolism exhibits a longer fluorescence lifetime compared to its free-floating form in glycolysis. Therefore, we can detect changes in proportions of NADH dedicated to certain metabolic activities by looking at proportions of fluorescence lifetimes in healthy vs. cancerous cells over time. To investigate this, SKH1 mice were exposed to UV radiation for 5 days a week and imaged at specific UV doses using FLIM to examine changes in cellular metabolism and collagen structure over time. In our newest experiment, all mice exposed to UV radiation for the full 25 week experiment developed skin lesions, and 11 out of 12 developed tumors. This was significantly improved over our previous experiment, which observed two tumors and ~50% lesion rate. We examined changes in tissue morphology using H&E staining to measure differences in skin thickening and cancer. UV treated mice showed significantly increased epidermal thickness, which was observed in both experiments. Epidermal thickness also differed between males and females. When analyzing changes in NADH conformation, we observed a significant difference in NADH protein bound fractions between UV and non-UV treated mice as well as between males and females. In addition, progression towards cancer development was marked with conspicuous changes in collagen structure. Collagen strands began to exhibit more erratic, unorganized patterns and appeared much shallower in the epidermis in lesioned areas, competing with cells for space. In conclusion, cancer development led to a clear, distinguishable development that was identifiable when compared to healthy cells. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
EFFECT OF CURCUMIN ON EXPRESSION OF GROWTH AND SURVIVAL GENES IN TRIPLE NEGATIVE BREAST CANCER
Emmanuella Tchona1, Isioma Akwanamnye1, and Ann Marie Buchmann1,
emmanuella.tchona@eagles.csc.edu
1- Department of Physical and Life Sciences, Chadron State College, Chadron, NE.
Triple negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cells. Known as triple negative because of the lack of expression of estrogen and progesterone and lack of overexpression of the human epidermal growth factor (HER2) receptors, it is diagnosed late, has a faster growth rate, limited treatment options and worse outcomes. Previous experiments with curcumin, the active ingredient in turmeric, have demonstrated its anti-inflammatory and tumor suppressing effects. The work done in our labs shows that curcumin inhibits breast cancer growth in cell lines MDA-MB231, causing them to go into apoptosis. Novel therapeutic approaches in our lab aim to target pro-growth and pro-survival proteins that play a role in the cell cycle and triple negative breast cancer by affecting the growth rate observed in TNBC cells. Our hypothesis is that curcumin may inhibit the expression of pro-growth and pro-survival genes. To test this hypothesis, we are examining the expression of genes regulated by the NFκB pathway. Using q-PCR we are quantifying changes in the expression of the following transcriptional targets of the NFκB pathway: Cyclin D-family members, Bcl-family members, BIRC5 and c-Myc. Results from past experiments show that the expression levels of BIRC5 and c-Myc at concentrations (2.5μM,5μM,10μM & 20μM) over a period of 24hrs, were decreased compared with those of the control group. Current directions include reproducing those results at different timepoints and over a wider range of concentrations.
INVESTIGATING THE ROLE OF HCMV US18 IN VIRAL INFECTION
Mary Helms1, Douglas Christensen1, and Lindsey B. Crawford2, mahelm01@wsc.edu
1 - Department of Life Sciences, Wayne State College, Wayne, NE;
2 - Nebraska Center for Virology, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE.
Human Cytomegalovirus (HCMV) is a beta-herpes virus which infects 44-99% of the population and establishes a lifelong infection due to its ability to avoid the immune system through periods of latency and reactivation. Though often undiagnosed, HCMV is the leading cause of infectious birth defects and can cause life threatening issues in patients who are immunocompromised. A better understanding of the processes of latency and reactivation is imperative to advancement of HCMV treatment and prevention. The gene US18, of the US12 gene family, is expected to code for a G protein-coupled receptor and has been shown to be expressed during latency. Based on this information, the focus of this research is to study US18 expression in human cell lines using amplification, cloning, and transfection into HEK-293 cells. Through this, we expect to better understand the role of US18 in viral infection and processes of latency and reactivation. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
MORNING SESSION-D2
UTILIZING A COMPREHENSIVE BACTERIAL SCREENING PANEL TO IDENTIFY NOVEL ANTI-MICROBIAL COMPOUNDS
Jeff Zimmerly1, Nicholas Armstrong1, Clare Euteneuer1, Yunos Alizai1, and Paul H. Davis1, jzimmerly@unomaha.edu
1 - Department of Biology, University of Nebraska at Omaha, Omaha, NE.
The rise of antibiotic resistance has created significant challenges in the treatment of once-curable infections. In 2019 alone, 1.27 million deaths were directly attributed to antibiotic resistance globally. Additionally, many current medications are accompanied by undesirable side effects and toxicity. To address these issues, we have developed a compound screening assay to identify novel antibiotics with potency against a diverse range of bacterial species representing human pathogens. The panel is composed of 5 bacteria including Proteus mirabilis, Neisseria mucosa, Listeria innocua, Mycobacterium smegmatis, and Staphylococcus epidermidis. Our screening assay has successfully identified several promising antibacterial compounds while demonstrating limited toxicity to human cells. Future efforts will focus on advancing the most potent, broadly acting compounds into in vivo studies. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
COMBATING ANTIMICROBIAL RESISTANCE UTILIZING OPENTRONS TECHNOLOGY
Nicholas Armstrong 1, Jeffrey Zimmerly1, Yunos Alizai 1, and Paul H. Davis 1, nicholasarmstrong@unomaha.edu
1 - Department of Biology, University of Nebraska at Omaha, City, NE.
New antimicrobial agents are needed to combat the rising antimicrobial resistance, making time of the essence. To reduce the amount of time it takes to develop new antimicrobial compounds we have leveraged the OpenTrons OT-2 robot to reduce data generation time and improve data quality. The utilization of the OT-2 Robot has reduced plating times from 50 minutes to only 20 minutes. In The future we plan to further optimize the screening process by making our compounds with the robot. Altogether, the precision of robotics can accelerate the screening process, increasing the quantity and quality of HITS for further anti-microbial development.
EXPLORING THE ROLE OF INLB FOR USE AS A DRUG DELIVERY AGENT
Jason Franklin1, Shawn Pearcy Ph.D.2, and Douglas Christensen Ph.D.2, jafran05@wsc.edu
1 - Department of Physical Sciences and Mathematics, Wayne State College, Wayne, NE;
2 - Department of Life Sciences, Wayne State College, Wayne, NE.
Listeria monocytogenes is a pathogenic Gram-positive bacillus-shaped bacteria that causes listeriosis infection. Listeriosis affects about 1,600 people annually, resulting in an average of 260 deaths per year, frequently as a result of the consumption of contaminated foods. A surface protein called Internalin B (inlB) promotes bacterial entry into mammalian cells, where the organism then escapes the phagosome, allowing the bacterium to flourish in the host cell cytoplasm. The ability of inlB to trigger phagocytosis may be useful as a conjugated drug delivery agent. Here we clone, verify and over-express inlB in E. coli in an attempt to purify the protein from inclusion bodies. Successful purification will aid in assessing biological activity of inlB in the absence of it being membrane bound. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
POLYVALENT PYRIDINIUM- AND QUINOLINIUM-SUBSTITUTED TRIAZOLES AS ANTISEPTICS
Adam Burr1 and James T. Fletcher1, ajb48719@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE.
Quaternary ammonium compounds (QACs) composed of N-heterocycles such as cetylpyridinium chloride are common commercial disinfectants. Recently, click-derived 1,2,3-triazolium salts have been shown to display antiseptic properties in a substituent dependent manner. The aim of this study was to prepare both pyridinium-substituted 1,2,3-triazole QACs and quinolinium-substituted 1,2,3-triazole QACs, as well as polyvalent hybrid salts possessing both pyridinium or quinolinium and triazolium subunits, and to evaluate their respective antiseptic properties. Under mild conditions, N-benzylation at either single nitrogen-containing heterocycle subunit could be achieved selectively over N-benzylation at the 1,2,3-triazole subunit. Under forcing conditions, N-benzylation at both single nitrogen-containing heterocycle subunits and 1,2,3-triazole subunits to form a hybrid polyvalent QAC was achievable. When comparing the ability of bis-triazole, para-phenyl bridged bis-triazole and para-biphenyl bridged bis-triazole analogs, the efficiency of preparing polyvalent hybrid triazolium products was distance-dependent with respect to the other heterocycle. Monovalent and polyvalent QACs were evaluated for antiseptic properties against exemplary Gram-positive bacteria (S. epidermidis and B. subtilis), Gram-negative bacteria (E. coli and K. aerogenes), and yeast (C. albicans and S. cerevisiae) using minimum inhibitory concentration assays. Micromolar MIC values were observed for select analogs, with overall charge as well as peripheral substituent identity influencing antiseptic potency. Details regarding the synthesis of target compounds, examination of relative N-benzylation rates and evaluation of antiseptic potency will be presented. This publication was made possible by grants from the National Institute for General Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
DENTAL CARIE RESISTANCE AND THE APPLICATION OF SEALANTS.
Jamie Graff1 and Rachelle Rider2, Jamie.graff@eagles.csc.edu
1 - Department of Physical Sciences, Chadron State College, Chadron, NE;
2 - Department of Life Sciences, Chadron State College, Chadron, NE.
Dental Caries are a commonly seen issue in the oral health of almost all individuals. Also known as cavities, dental caries occur when a combination of acid-producing bacteria and the fermentation of carbohydrates takes place in the presence of saliva and left-over foods. Cavities are prevalent in the primary teeth of children due to the weak outer coating of the teeth, also known as enamel. Cavities form under various conditions including the presence of cariogenic bacteria, lack of fluoride exposure, or lack of oral care. Methods used to prevent dental caries from forming include brushing teeth regularly, fluoride rises, and the application of sealants. Sealants are a UV-light cured resin that is applied to the chewing surfaces of teeth. Once applied, the teeth are temporarily protected from decay. Sealants are commonly placed as a precaution to potential decay, but are they worth the money? In a laboratory setting, Sealants were placed on bovine teeth and exposed to vinegar to determine the resistance to erosion over time. The teeth will come in contact with the vinegar solution for four hours for 30 days. Results are not yet finalized, but at the conclusion of the trial, a scanning electron microscope and photographic images will determine the amount of sealant and tooth erosion.
AFTERNOON SESSION-A3
ABC TRANSPORTER MODULATING RESPONSES TO LLINS FOR MALARIA MOSQUITOES
Heather M. Hernandez1, Leslie C. Rault1, Troy D. Anderson1, hhernandez3@huskers.unl.edu
1- Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE
Malaria is a life-threatening disease caused by Plasmodium spp. transmitted to humans through the bite of infected female Anopheles spp. mosquitoes. The reduction of mosquito population densities is the primary strategy to mitigate the community transmission of malaria. The use of larvicides, indoor residual insecticide sprays, and long-lasting insecticidal nets (LLINs) are recommended and effective tools to reduce the population densities of malaria mosquitoes. The mosquito central nervous system (CNS) is a proven target site for high efficacy insecticides. However, due to continued use, widespread resistance and inefficient target-site delivery has threatened this approach, reducing the efficacy of LLINs. Thus, insecticide resistance and target-site delivery are serious public health challenges that warrant the development of improved chemical interventions for mosquito populations and disease transmission. ATP-binding cassette (ABC) transporters are integral membrane proteins at the mosquito blood-brain barrier interface acting as molecular “vacuum cleaners” to traffic out insecticides and reduce the intracellular delivery of these chemistries to their intended target sites. The data presented here will provide evidence of a blood-brain barrier obstacle for the intracellular delivery of insecticides to the mosquito central nervous system. A series of chemistries that target ABC transporters will be reported to modulate the efficacy of long-lasting insecticidal nets (LLINs) to Anopheline mosquitoes. These data will be discussed as an approach to identify conventional insecticides as substrates for ABC transporters and to screen drug libraries for ABC transporter inhibitors that can be used as an alternative chemical intervention for Anopheline mosquitoes.
PHAGOSTIMULANT ACTIVITY OF AMINO ACIDS TO IMPROVE NEXT-GENERATION ATTRACT-AND-KILL TECHNOLOGIES
Xi Xian Ng1, Ellis J. Johnson1, Leslie C. Rault1, Troy D. Anderson1
1 - Department of Entomology, University of Nebraska, Lincoln, NE USA
Email address : xi-xian.ng@huskers.unl.edu
The incidence of mosquito-borne diseases is a significant threat to human health with effective chemical interventions limited by widespread insecticide resistance. There is evidence for attract-and-kill technologies as promising interventions to reduce mosquito populations and interrupt community transmission of diseases. The longevity of mosquitoes to rest and feed on attract-and-kill chemical compositions is a challenge that compromises the efficacy of these interventions to reduce mosquito populations. This study examined phagostimulants to enhance the resting and feeding activity of mosquitoes and, in turn, increase toxicant ingestion leading to higher mortality of mosquitoes. Here, we present the spatial attraction, locomotor activity, olfactory response, feeding rate, and survivorship of Aedes aegypti presented with amino acid additives to chemical compositions for use in next-generation attract-and-kill technologies.
IMPROVING AN OPEN-SOURCED AUTOMATED MICROPLATE ASSAY FOR ANTI-INFECTIVE AND CYTOTOXIC COMPOUND SCREENING
M. Yunos Alizai1, Brianna N. Davis1, Paul H. Davis1 yalizai@unomaha.edu
1 - University of Nebraska at Omaha, Omaha, NE.
Microplate assays are important tools for anti-infective compound screening, cytotoxicity screening, and drug discovery. These microplate assays provide valuable insights into the efficacy of anti-infective compounds and their potential for use in the drug discovery process. These assays help determine the effectiveness of a substance or compound against pathogens and information on the potential toxicity of a substance or compound by measuring impacts on host cell metabolism, aiding in the evaluation of their suitability for clinical applications in combating infections. However, these assays take time and suffer from user-associated variations when testing multiple compounds for multiple pathogens and host cells. The development of high-throughput screening methods for compound assays is essential for the rapid evaluation of potential compounds in the drug discovery process. In this study, we describe the development process of an automated wide-spectrum screening assay for anti-infection compounds assay utilizing the OpenTrons robot. This assay can process a large number of samples in a reduced period of time and the automation reduces human error. To date, our findings support the feasibility of using the OpenTrons robot for an automated compound screening assay, providing a valuable tool for the drug discovery process. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
IDENTIFICATION OF STAPHYLOCOCCUS LUGDUNESIS PROTEASES THROUGH ETHYL METHANESULFONATE MUTAGENESIS
Caleb Rother1, Justine Pitzer1, Austin Nuxoll1, rotherc@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Staphylococcus lugdunensis, like Staphylococcus aureus and Staphylococcus epidermidis, is common in the human skin flora. Despite S. lugdunensis causing aggressive and often hard to treat infections, the pathogenesis of this organism remains largely understudied. Numerous proteases have been connected to pathogenesis and immune evasion strategies in the closely related pathogens, S. aureus and S. epidermidis. To identify proteases that S. lugdunensis employs in these processes, we designed a high-throughput screen following ethyl methanesulfonate (EMS) mutagenesis. These mutants were then separated using a cell sorter and grown in a 96 well plate. Using this library, we screened for differences in proteolytic activity in comparison with wild type S. lugdunensis. The library of EMS treated S. lugdunensis was grown for 48 hours at 37℃ before plating on a blood-agar plate. After 48 hours of incubation at 37℃, proteolytic activity was determined by a zone of clearing. Mutants exhibiting decreased proteolytic activity will be confirmed and growth kinetics will be assessed for growth defects. Individual mutants will then be sequenced and analyzed to determine mutations responsible for protease activity. Further studying of S. lugdunensis proteolytic activity offers important insight into the pathogenesis of this organism. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
EFFECTIVE ISOLATION AND IDENTIFICATION TECHNIQUES OF HSP70 IN INSECT POPULATIONS
Savannah Armendariz1 and Dr. Jennifer Grove,
1- Department of Biology, College of Saint Mary, NE.
Heat shock proteins (hsp) are necessary molecular chaperones that are involved in numerous life functions in all living organisms. More importantly, the expression of these proteins varies depending on the environmental factors and developmental differences. There are many types of heat shock proteins but Hsp70 is present in many eukaryotic organisms and plays a key role in protein metabolism. In humans, Hsp70 is produced in response to stress. In response to stress, Hsp70 will bind to protein substrates to prevent denaturation or aggregation. Our primary purpose currently, will be to determine the most effective technique for isolation and detection of Hsp70 in insect samples. This study will initially utilize fly larvae at their third instar stage of development. The hsp expression will be upregulated by heat induction at a variety of temperatures for 15 minutes and then fixed and stored in ethanol for protein isolation. Protein extraction will be followed with immunoblotting for the Hsp70 using commercially purchased antibodies. Other stages of fly development will also be heat induced to use for protein isolation and detection. Certain species of insects have adapted to extreme environments including extreme temperatures. Cicindelidia haemorrhagica, commonly known as the Wetsalts Tiger Beetle, have adapted to withstand the extreme conditions of the hot springs and geysers found in Yellowstone National Park. The hot springs and geysers are inhabited by a variety of thermophilic organisms that evolved to survive the extremely hot temperatures. Outside of Yellowstone, C. haemorrhagica primarily inhabits moist, saline flats in the Western United States. Due to the presence of C. haemorrhagica in Yellowstone, it is theorized that the species has evolved to develop an increased heat resistance to live in the extreme conditions of the hot springs and geysers. In these extreme conditions, the species has shown to have a decreased internal body temperature and exhibit less cooling behaviors compared to those living outside of Yellowstone. Although these findings are indicative that the species has evolved to withstand the harsh conditions associated with Yellowstone, there are no indefinite biological adaptations yet found. After identifying the most effective technique for Hsp70 identification, we plan to isolate and detect Hsp70 from Wetsalts Tiger Beetle samples collected from multiple locations found within Yellowstone National Park. Data will be presented. This project is funded by INBRE.
AFTERNOON SESSION-B3
QUANTIFICATION OF NADH PHASOR FLIM AND P53 EXPRESSION FOR UV-INDUCED SKIN CANCER IN VIVO
Greer L. Porter1, Alex Chen1, Kennedy A. Haase1, Reese Kolar 1, Jackson M. Laurent1, Jonathan Li1, Aidan O’ Mara1, Maimuna Olow Nagey1, Jalen K. Ramos1, Derek A. Remitar III1, Abraham J. Saks1, Hannah Schloman1, Jinann A. Shoshara1, Zachary J. Smith1, Fiona Sun1, Jacob A. Sweet1, Jake S. Wakahiro1, Laura A. Hansen2, Michael G. Nichols2 , greerporter@creighton.edu
1 - Department of Physics, Creighton University, Omaha, NE;
2 - Department of Biomedical Sciences, Creighton University, Omaha, NE.
Two key signs of cancer progression include significant changes in both metabolism and tissue architecture, but these changes are often discovered late in the diagnosis of certain cancers. To investigate a more efficient, non-invasive method of diagnosing skin cancer, in vivo metabolic changes of the epidermis and dermis were quantified through fluorescence lifetime imaging microscopy (FLIM) of NADH while biochemical responses were analyzed through immunofluorescence of tumor suppressor p53. A total of 30 SKH1 mice were used for a preliminary study in which we observed chronic UV- and sham-treated SKH1 mice over a period of 26 weeks. Images of the epidermis and dermis were taken biweekly at a minimum of four locations at different depths throughout the epidermis and dermis. Through FLIM, the time-resolved NADH fluorescence decay was measured, and phasor sine and cosine transformations were used to calculate the free:protein-bound ratio of NADH. This ratio was used to monitor changes in metabolic activity. Significant changes in the NADH bound fractions in UV-treated mice were observed in comparison to sham controls, with the most pronounced change in tumor-bearing female mice. Immunofluorescence of p53 was performed to identify UV-induced DNA damage and tumor progression and confirm our findings with FLIM. Preliminary data suggests an upregulation of p53 within the UV-treated mice in comparison to sham controls. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
INVESTIGATING METABOLIC CHANGES UNDER NORMOXIC AND HYPOXIC CONDITIONS THROUGH NADH PHASOR FLIM IN VITRO WITH INTEREST IN DEVELOPMENT OF A NON-INVASIVE DIAGNOSTIC BIOPSY IN VIVO
Abraham J. Saks1, Jackson M. Laurent1, Elizabeth M. Cronin1, Greer L. Porter1, Derek A. Remitar1, Aidan J. O’Mara1, Jinann A. Shoshara1, Reese A. Kolar1, Kennedy A. Haase1, Laura A. Hansen2, Michael G. Nichols1, ajs13675@creighton.edu
1 - Department of Physics, Creighton University, Omaha, NE;
2 - Department of Biomedical Sciences, Creighton University, Omaha, NE.
In the US, 20% of all non-melanoma skin cancer cases are squamous cell carcinoma (SCC). Cutaneous SCC is attributed as being metastatic with around 1-5% of cSCCs metastasizing. These characteristics can be explained by metabolic and structural changes. The Warburg Hypothesis (1913) describes a shift in proliferation from oxidative phosphorylation towards glycolysis in cancerous cells. Additional changes in metabolism are observed with the expression of human epidermal growth factor receptor 2 (HER2). Fluorescence lifetime imaging microscopy (FLIM) of NADH was performed to produce a protein-bound: free ratio to monitor metabolic changes. SCC 74A (low HER2) and SCC 74B (high HER2) cell lines were cultured in normoxic (21% O2) and hypoxic (<2% O2) environments to quantify changes in metabolism using NADH Phasor FLIM microscopy. We tested the hypothesis that prolonged growth in a hypoxic environment would result in a shift from aerobic to anaerobic glycolysis, with less reliance on the electron transport chain (ETC). In addition, we tested the effect of HER2 expression on ETC activity, evaluated by mitochondrial inhibitors and uncouplers, under both environmental conditions. A three-way (cell line, treatment, oxygen availability) ANOVA revealed a significant decrease in the fraction of protein bound NADH and utilization of the electron transport chain when SCC74B cells were cultured under hypoxic conditions. Inhibition of HER2 caused a similar decrease. Taken together, NADH Phasor FLIM was able to non-invasively reveal metabolic changes associated with both of these factors in vitro. Continuing with these results, new noninvasive, optical methods of biopsy can be created to diagnose cancer. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
REGULATION OF VLDLR GENE EXPRESSION BY MICRO RNA-100 IN MCF-7 HUMAN BREAST CANCER CELLS BEFORE AND AFTER THE EPITHELIAL TO MESENCHYMAL TRANSITION
McKenna Revis1, and Kate Marley1, mckenna.revis@doane.edu
1 - Department of Biology, Doane University, Crete, NE.
Each year more than 200,000 men and women are diagnosed with breast cancer and more than 40,000 people die from breast cancer. Of women born in the United States approximately 1 in 8 are expected to develop breast cancer in their lifetime. When breast cancer is diagnosed at an earlier stage, before it metastasizes, clinicians are more successful at treating and eliminating the cancer. In fact, over 90% of cancer deaths are due to metastasis. Most human cancers develop in epithelial tissues, but as tumors grow, they can accumulate mutations that lead to cells adopting more mesenchymal tissue characteristics and gene expression profiles. Tumors that have undergone this epithelial-to-mesenchymal transition (EMT) are more motile and aggressive and prognosis for patients is poorer. Understanding the changes in gene expression that underlie the EMT may provide opportunity for future therapies to prevent metastasis or treat more aggressive cancers. Some of these changes are thought to be controlled by micro-RNA regulation of gene expression. In particular, micro-RNA 100 (miR-100) has been identified as a micro-RNA that reduces translation of mRNAs whose functional protein prevents EMT, in part. The gene for Very Low Density Lipoprotein Receptor (VLDLR) has been shown to have a 3’UTR sequence complementary to miR-100 suggesting it could be a target gene regulated by miR-100. We have collected total RNA from MCF-7 epithelial human breast cancer cells and MCF-7M mesenchymal human breast cancer cells derived from MCF-7 cells passaged through at least 5 continuous rounds of tumorsphere culture. We will present our analysis of VLDLR gene expression in MCF-7 and MCF-7M cells before and after EMT. Women diagnosed with breast cancer while it is still localized in one tumor are 99% likely to survive their diagnosis, however once breast cancer metastasizes to distant tissues, five-year survival rates decrease to 22%. Understanding the gene expression profile involved in EMT and metastasis is a major part of strategies to effectively treat these more aggressive cancers.
EFFECT OF HIGH GLUCOSE ON FOCAL ADHESION AND RHO KINASE IN BREAST CANCER CELLS
Payton Sindelar1, and Surabhi Chandra1, sindelarp@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Chronic diabetes exacerbates several health conditions including cancer. Concomitant diabetes and breast cancer have been associated with high mortality. Our lab has previously shown that polyamine pathway is involved with the proliferation of breast cancer cells, however specific enzymes involved in this pathway have not been fully investigated. Moreover, phytochemicals such as black seed oil (BSO) have been shown to have cytotoxic potential in certain cancers but its role in diabetic breast cancer has not been explored yet. The hypothesis of this study was that cytoskeletal modulating enzymes focal adhesion kinase (FAK) and Rho kinase are downregulated in high glucose conditions in breast cancer cells, and this effect can be prevented using BSO. Triple negative breast cancer cells, MDA-MB-231 and estrogen positive cells MCF-7, were used for the study and treated with varying concentrations of glucose and BSO. Western blot technique was performed for protein analysis. It was observed that the protein levels of FAK and Rho kinase (ROCK1 and ROCK2) fluctuate with high glucose treatments. Experiments in combination with BSO prevented any changes in the levels of FAK and Rho kinase. Since these enzymes are involved with cell structure and function, it is likely that they can affect cell metastasis as well, and BSO can be protective in these conditions.
AGGRESSIVE PROLIFERATION IN ADVANCED PROSTATE CANCER
Kaitlin Smith1,2, Avery Stillahn1, Sreyashi Bhattacharya1, Samikshan Dutta1, Kaustubh Datta1 ksmith7907@csm.edu
1 - UNMC Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE;
2 – Department of Biology, College of Saint Mary, Omaha, NE.
Prostate cancer is the second leading cause of cancer death in men in the United States. It originates in the luminal cells of the prostate, the most abundant cell type in the gland. The initial progression of prostate cancer is measured by the levels of prostate-specific antigen (PSA). Hormonal therapy to reduce testosterone levels often provides an effective initial treatment. The prostate cancer transitions into castration-resistant prostate cancer (CRPC), where even under low levels of testosterone, the cancer cells still survive, and patients then undergo androgen deprivation therapy. However, 20-30% of CRPC patients subsequently develop therapy-induced neuroendocrine-like prostate cancer (t-NEPC), which is difficult to diagnose and treat due to nonspecific biomarkers. T-NEPC cells undergo specific changes to evade treatment, including genetic, epigenetic, and pathologic adaptations. This results in the formation of tight cell clusters and increased aggression. This research aims to understand the proliferation mechanisms of neuroendocrine-like prostate cancer cells. We hypothesized that these neuronal adhesion molecules are major components for cell proliferation in t-NEPC cells.
This study uses LNCaP C4-2 "DKD" cells and LNCaP C4-2BER cells, which are human prostate cancer cell lines that have been previously isolated and have neuroendocrine phenotypes. The study found that NrCAM, a neuronal cell adhesion molecule, is upregulated in t-NEPC cells and promotes proliferation. Knocking down NrCAM in these cells reduces their ability to adhere together and stabilize their cytoskeleton, which in turn reduces their ability to proliferate. The DKD cells had a significant increase in their cluster length, and immunofluorescence tagging cancer proliferation factor Ki67 showed that the knockdown of NrCAM and the addition of doxycycline reduced the proliferation of these cells. Additionally, immunofluorescence tagging phalloidin using C4-2BER cells showed that the depletion of NrCAM disorganizes the cytoskeleton of the cells.
The results of the study suggest that NrCAM plays a key role in the proliferation of t-NEPC cells and that targeting this molecule could be a potential strategy for treating this aggressive form of prostate cancer. Future research will use in vivo mouse models to further investigate the distribution and behavior of t-NEPC cells and explore the use of MK801, a drug that binds to glutamate receptor and acts as an inhibitor of glutamate binding, to reduce the proliferation of t-NEPC cells.
"The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.”
AFTERNOON SESSION-C3
EXPLORING THE INTERACTION OF HCMV UL5 WITH HOST CELLULAR GENES ANKRD13A AND ST3GAL1
Kai Waddell1,2, Kamryn Pfenning2,3, and Lindsey Crawford2,3, kwaddell2@huskers.unl.edu
1 - Department of Microbiology, University of Nebraska-Lincoln, Lincoln, NE;
2 - Nebraska Center for Virology, Lincoln, NE;
3 - Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE.
Human Cytomegalovirus (HCMV) is a common β-Herpesvirus with a seroprevalence of about 60-90% among the general population. It remains a significant pathogen due to its latent, lifelong infections and significant complications in those pregnant or immunocompromised. To understand how this virus causes disease and persists, we are studying the function of a viral gene, UL5, with unknown function. To begin, we are determining the role of HCMV UL5 during lytic replication, a specific viral life cycle stage where the virus is active and directly producing new virus particles. A prior proteomics study indicated that UL5 may directly interact with cellular proteins ANKRD13A and ST3Gal1. Both cellular proteins are important for protein regulation and immune function which are key pathways dysregulated by HCMV. We determined that HCMV infection upregulates ANRKD13A and ST3GAL1. Subsequently, we transfected cells with plasmid constructs containing UL5 to study its expression and cellular interactions individually. We found ANKRD13A and ST3Gal1 were upregulated in UL5-transfected cells. We are continuing to explore the direct interaction of UL5 with ANKRD13A and. ST3GAL1 at the protein level. Finally, we have preliminary data that suggests UL5 is expressed during latency, another critical part of HCMV’s life cycle. Our future studies will continue to characterize the role of HCMV UL5 in lytic and latent infection of HCMV to advance our understanding of HCMV infection and latency and open avenues for targeted therapeutic interventions.
THE PURSUIT OF PYRUVATE DEHYDROGENASE KINASE INHIBITORS TO TREAT METABOLIC DISEASE
Noah Shackelford1, Allen A. Thomas1, and Michael A. Moxley1, shackelfordn@lopers.unk.edu
1 – Department of Chemistry, University of Nebraska at Kearney, Kearney, NE.
The pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that links the central metabolic pathways of glycolysis and the tricarboxylic acid cycle, and is regulated in part by its inhibitor pyruvate dehydrogenase kinase (PDK). Overexpression of PDK, and therefore decreased activity of PDC, is associated with numerous metabolic disorders including various forms of cancer, type 2 diabetes, and heart disease. This project involved the virtual screening of 15 million compounds from the ZINC database against the lipoamide binding site of isoforms PDK 1-4. This search yielded eight compounds sharing a common biphenyl structure and high binding scores for the four isozymes of PDK found in humans. Of the eight compounds, six were synthesized in house, and assayed via absorbance associated with PDC activity. Two compounds inhibited PDK, restoring PDC activity in a concentration-dependent manner. Further research into these compounds and related structures is ongoing, with the ultimate goal of yielding compounds that can be used to treat metabolic disease.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
CHARACTERIZING AN ESSENTIAL AMINO ACID REQUIRED FOR NUCLEOSOME ASSEMBLY
Emma Foley1 and Lynne Dieckman1, eef85459@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE.
When a cell divides, its genome must be replicated and passed on to the next generation of cells. During replication, newly synthesized DNA is organized into nucleosomes, the fundamental units of chromatin. Proliferating cellular nuclear antigen (PCNA) and chromatin assembly factor 1 (CAF-1) are two proteins required for this process. The interaction between PCNA and CAF-1 is essential for nucleosome assembly to occur. However, the mechanism of this interaction is not well understood. Our lab has discovered an amino acid in PCNA, R44, that resides in a potential novel interaction site for CAF-1. I performed protein-protein binding studies between R44A PCNA and CAF-1 or other PCNA-interacting proteins and determined that the R44A mutation results in increased binding in some but not all PCNA-binding proteins. These studies provide insight into a potentially inhibitory role of R44 in PCNA, which highlights the importance of this residue in the maintenance of DNA organization and genome stability.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
EVALUATION OF RESIDUAL PRIONS FROM DISINFECTED PRION-CONTAMINATED SURFACES UTILIZING RT-QUIC.
Vivianne Paine1, Jason Bartz2 , jbartz@creighton.edu
1- College of Saint Mary, Omaha, NE;
2 - Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE.
Prions are abnormal pathogens causing prion diseases. Prions are misfolded cellular prion proteins (PrPc) where the misfolding can be spontaneous, inherited, or induced by interacting with the pathogenic form (PrPSc). Iatrogenic and occupational transmission of human prion disease (Creutzfeldt-Jakob disease) has been documented via incompletely disinfected surgical equipment and/or other prion-contaminated sources. Therefore, a rapid and reliable methodology to evaluate prion disinfection sufficiency of relevant laboratory and medical surfaces is in critical need. This study investigated the applicability of using the previously developed swabbing-RT-QuIC technique to evaluate the disinfection efficiency of prion-contaminated surfaces by measuring the RT-QuIC seeding activity of surface-recovered residual prions through swabbing and sonication-extraction. Experiments were conducted on stainless steel (304 grade) tokens. First, we determined the detection sensitivity for surface-recovered prions. We prepared a 10-fold serial dilution of HY TME prions (a hyper strain of hamster-adapted transmissible mink encephalopathy) and applied them from each dilution to the tokens. Prions were recovered from the tokens after drying (> 99% of water loss) followed by RT-QuIC analysis. We found an approximate one-log reduction in the detection of surface-recovered HY TME when compared to the controls (the 10-fold serial dilution of HY TME not undergoing surface drying and recovering). Next, we quantified the disinfection efficiency for HY TME prions from contaminated surfaces with different detergents. We applied HY TME prions from -2 and -4 (log) dilutions to the stainless-steel tokens and treated the surface-dried prions with ultrapure water (non-disinfection control), 70% ethanol, or undiluted bleach. Residual HY TME prions in the swab extracts were analyzed using an endpoint (10-fold serial dilution) detection with RT-QuIC. We determined that water and ethanol did not disinfect surface dried HY TME prions as the RT-QuIC titer was unchanged (< 1 log, p > 0.05) SD50 (the dilution at which 50% of replicates are considered infectious and are indicative of infectivity) compared to surface dried HY TME prions. In contrast, undiluted bleach significantly (p<0.05) reduced HY TME titer on the surface of the tokens by 6 logs of SD50. The results indicated the ineffectiveness of ethanol and the effectiveness of undiluted bleach in disinfecting dried HY TME prions on stainless steel surfaces. To expand the swabbing-RT-QuIC technique to other surfaces, we will test proprietary resin blocks representing laboratory bench tops following the same procedure as for stainless steel tokens.
EXAMINING SEX DIFFERENCES IN THE RESPONSE OF LUNG TYPE 2 INNATE LYMPHOID CELLS TO PEANUT
Alethia Henderson1, Leigh-Anne Lehmann1, Sunanda Rajput1, Nicholas J. Hobbs, Ph.D.1, and Joseph J. Dolence, Ph.D.1, hendersonaj@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Much remains to be learned about how peanut (PN) initiates immune responses to elicit PN allergy. Specifically, how sex differences impact the development of PN-specific immune responses remains unclear. We have shown that type 2 innate lymphoid cells (ILC2s) in female lungs display greater responses than those in males after 3 days of PN inhalation. This study aimed to further elucidate how ILC2s in the lungs of mice react to PN during the genesis of PN allergy. We exposed male and female mice to PN in an 11-day inhalation model to investigate how sex differences impacted the response of lung ILC2s at a time point just before PN-specific adaptive responses begin to elicit PN-specific IgE. Female mice showed a greater ILC2 response to PN compared to males in the lungs after 11 days of PN exposure. Furthermore, the ILC2s expressed IL-1R1 in response to PN suggesting ILC2s directly respond to IL-1 alpha released by lung epithelial cells after PN inhalation. Additionally, we are developing a protocol to visualize lung ILC2s using confocal microscopy. Future studies using both flow cytometric and microscopic approaches will enable us to further understand how sex differences impact activation of ILC2s against PN.
AFTERNOON SESSION-D3
EXPLORING CHANGES IN COACTIVATION, STRENGTH, AND GROSS DEXTERITY BEFORE AND AFTER AN 8-WEEK HOME INTERVENTION IN CHILDREN WITH UPPER LIMB REDUCTIONS
Liliana Delgado1 and Dr. Jorge Zuniga 1, lkdelgado@unomaha.edu
1 - Department of Biomechanics, University of Nebraska Omaha, Omaha, NE.
Conventional approaches to understanding prosthetic rejection have predominantly focused on extrinsic factors, overlooking critical neuromuscular aspects. This study addresses this gap by concurrently investigating coactivation, muscle strength, and gross dexterity before and after an 8-week home intervention in children with upper limb reduction deficiency. The research seeks to provide a comprehensive understanding of the physiological and functional changes associated with prosthesis use. Nineteen participants, aged 3-19, with congenital upper limb reductions, underwent an 8-week home intervention utilizing 3D-printed prostheses. Coactivation, strength, and gross dexterity were assessed before and after the intervention, employing EMG signals for coactivation, maximal voluntary isometric contractions (MVC) for strength, and the Box and Blocks Test for gross dexterity. Following the home intervention, there was a nonsignificant reduction in coactivation and a significant increase in muscle strength across all conditions. Gross dexterity improved in both affected and non-affected hands. Correlation analysis revealed a stronger inverse relationship between coactivation and strength post-intervention. These findings suggest that guided training at home positively impacts physiological adaptations, contributing to improved prosthesis acceptance. The observed enhancements in coactivation and strength lend support to the idea that practical training can refine motor control strategies, mitigating functional limitations. The study's innovation lies in its simultaneous assessment of coactivation, strength, and function before and after an 8-week home intervention, offering valuable insights into the intricate dynamics of prosthetic use in children. Further investigations with larger sample sizes are warranted to validate these findings and guide future interventions for pediatric prosthetic users.
POST-TRAUMATIC STRESS DISORDER SEVERITY FOLLOWING A TRAUMATIC EVENT MODULATION BY NEIGHBORHOOD-LEVEL FACTORS
Henry Drvol1 and Trey Andrews1, hdrvol2@huskers.unl.edu
1 - Department of Psychology, University of Nebraska-Lincoln, Lincoln, NE.
Post-traumatic Stress Disorder (PTSD) is a debilitating mental health condition that is onset by either experiencing or becoming a witness to a traumatic event. Learning models of PTSD hypothesize that stimuli that co-occur with traumatic events become associated with that event. This can include the location, the time, and other neutral contextual factors becoming associated with a traumatic event. Recovery following trauma may become more likely in a context where safety and danger are easier to predict accurately. It has been observed in prior research that neighborhood and context of an individual may modulate the onset of PTSD following a traumatic event. Learning models of PTSD as well as prior research show that the context of an individual could be associated with the likelihood to develop symptoms as well as the severity of those symptoms in individuals. There is a need to better understand if and how neighborhood factors, including violent crime rate, that could potentially be associated with PTSD. Following a visit to the emergency room, patients are given free resources and follow-up in order to see if PTSD symptoms develop and to treat these symptoms. Through follow up and gathering information about the neighborhood context of participants, the relationship neighborhood factors have with PTSD can be understood better.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
COMMUNICATION ERROR: RELATIONSHIP BETWEEN NEURAL CELL ADHESION MOLECULE EXPRESSION AND MUSCLE ACTIVATION IN PARKINSON’S DISEASE
Alexis Tonnemacher1, Mitchel Magrini1, and Kelley Hammond1, lexietonnemacher@creighton.edu
1 - Department of Exercise Science and Pre-Health Professions, Creighton University, Omaha, NE.
BACKGROUND: Neural cell adhesion molecule (NCAM) is expressed by myofibers when they are denervated and need reinnervation. If a myofiber fails to be reinnervated, it will undergo apoptosis which may influence muscle activation. However, the relationship between NCAM expression relative to myofiber type (rT1, rT2) and muscle activation is unknown. PURPOSE: The purpose of this pilot study was to investigate the relationship between relative NCAM expression and muscle activation in persons with Parkinson’s Disease (PD). METHODS: Muscle biopsies of the vastus lateralis were collected from six persons with PD (F=4; 67±9 yrs). Immunohistochemistry was used to analyze relative NCAM expression in PD. Muscle activation was measured using surface electromyography (EMG) of the vastus lateralis and torque was assessed during a maximal voluntary isometric contraction. Pearson correlation coefficients (r) were used to determine relationships between dependent variables. Significance was set at p<0.05. RESULTS: rT1/NCAM+ was negatively correlated with pRERn (r=-0.94, p=0.005) and pEMG (r=-0.88, p=0.02). rT2/NCAM+ was negatively correlated with peak torque (r=-0.96, p=0.002). There were no significant correlations between all other variables. CONCLUSION: Our pilot data suggests that greater rT1/NCAM+ expression is associated with a slower rate (i.e., pRERn) and reduced amplitude (i.e., pEMG) of muscle activation. Increased denervation of T2 myofibers (i.e., greater T2/NCAM+) is associated with decreased peak torque, suggesting a contractile consequence of NCAM expression. Overall, higher NCAM expression may be indicative of compromised communication between the nervous system and skeletal muscle.
THE POTENTIAL EFFECTS OF AN FDA-APPROVED ANTIDEPRESSANT TRAZODONE ON DYSLIPIDEMIA
Naara Ramirez1, and Yipeng Sui1, ramirezn@lopers.unk.edu
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could increase the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within the PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In the present research we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.
THE EFFECT OF PROPRIOCEPTIVE NEUROMUSCULAR FACILITATION ON BALANCE
Kayla Brown1 and Rachelle Rider2, kayla.brown1@eagles.csc.edu,
1 - Department of Physical Sciences, Chadron State College, Chadron, NE;
2 - Department of Life Sciences, Chadron State College, Chadron, NE.
On average, one out of every four elderly adults fall every year and almost ninety percent of athletes will get injured throughout their career due to lack of balance and mobility. Ability to hold up one’s weight and maneuver it is the ultimate deciding factor in whether a person will retain the ability to perform household tasks later in life or perform athletically in a sport. Balance ability must be practiced to retain the skill. Another method to retain balance is to expand the persons’ usable range of motion (ROM) through stretching. Proprioceptive neuromuscular facilitation (PNF) has been proven to be an effective form of stretching that improves both ROM and overall mobility. Contract-relax-antagonist-contract (CRAC) is the subset of PNF found to be the most effective in increasing ROM and mobility, making it the most viable subset to study. Using a force plate, a single leg standing test was performed both before and after PNF procedure to analyze balance ability of selected participants. CRAC procedure was performed on the target sural muscles, and antagonist crural muscles in a repetitive pattern for a short period of time. Balance patterns between pre- and post-CRAC procedures were analyzed and yielded little to no quantifiable difference in participants’ balance ability.
AFTERNOON SESSION - A4
EXPLORING THE INFLAMMATION REGULATORY FUNCTION OF LONG NON-CODING RNA EXPRESSION IN AMINOGLYCOSIDE INDUCED OTOTOXICITY
Collin Jackson1, Emily Daffer1, Tian Cong2, Peter Steyger2, and Annemarie Shibata1, cmj36688@creighton.edu
1 - Department of Biology, Creighton University, Omaha, NE;
2 - Department of Biomedical Sciences and Translational Hearing Center, Creighton University, Omaha, NE.
Aminoglycoside antibiotics are frequently prescribed in clinical practice because of their effectiveness and relative low cost when treating infection. Ototoxicity and permanent hearing loss has been recorded in 20-50% of patients with bacterial ear infections that are treated with aminoglycosides. This damage is exacerbated by infection-induced inflammatory responses in the cochlea. To develop novel treatments for the alleviation or inhibition of these inflammatory responses associated with ototoxicity, we must better understand the molecular mechanisms behind pro-inflammatory pathways in the inner ear. Preliminary data show that inflammatory responses in both animal models and in auditory cell lines involve regulation of gene expression by long noncoding RNA (lncRNAs). Our in vitro data demonstrate that differential expression of lncRNAs in House Ear Institute of Corti 1 (HEI-OC1) as well as Mouse Distal Convoluted Tubule (MDCT) cells occurs following exposure to bacterial lipopolysaccharide (LPS). Additional cell line work indicates differential expression of lncRNAs and cytokines after stimulation of cells with the aminoglycoside Kanamycin. Data suggest that lincRNA-Cox2 and lincRNA-Tnfaip3 are significantly upregulated when auditory cells respond to LPS as compared to controls and both lncRNAs are significantly downregulated when exposed to aminoglycoside treatment. I hypothesize that differentially expressed lncRNAs, like lincRNA-Tnfaip3 and lincRNA-Cox2, play a role in the regulation of gene transcription downstream of TLR4 receptor signaling. To test this hypothesis, in vitro and in vivo model systems are used. Toll-like Receptor 4 (TLR4), Myeloid Differentiation primary response 88 (MyD88), and Transient Receptor Potential Vanilloid 1 (TRPV1) knockout C57BL6/nJ mice and C57BL6/nJ wild type male and female mice were intraperitoneally injected with LPS and euthanized 24 hrs postinjection. Mice injected with HBSS served as controls. RNA from brain and brain stem of LPS-injected and control mice was used for RT-qPCR analyses. The knockout of TLR4 and MyD88 led to ubiquitous down regulation of lincRNA-Tnfaip3 and LincRNA-Cox2 in mouse brain and brain stems compared to LPS stimulated controls. In vitro experiments were used to examine the effect of silencing lincRNA-Cox2 on the expression of proinflammatory cytokines. Silencing of lincRNA-Cox2 resulted in a ~2fold decrease in the expression of Ccl2 and IL-6 relative to Gapdh after 1hr of LPS stimulation. A more robust understanding of the mechanisms of inflammation in the cochlea and the mechanisms of ototoxicity could identify therapeutic targets for the treatment or prevention of hair cell death and hearing loss following antibiotic delivery for ear infections. Given the number of people treated globally with aminoglycosides each year, there is a clear and present need to understand the mechanisms of aminoglycoside-induced ototoxicity. This publication was made possible by grants from the National Institute for AIDS and Infectious Disease (NIAID) (1 R15 AI156879) and the National Institute for General Medical Science (NIGMS) (5P20GM103427), components of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS or NIH.
DETERMINING EXPRESSION OF LNCRNAS IN AMINOGLYCOSIDE INDUCED OTOTOXICITY
Emily Daffer1, Collin Jackson1, Tian Cong2, Peter Steyger2, and Annemarie Shibata1 ERD52434@creighton.edu,
1 - Department of Biology, Creighton University, Omaha, NE;
2 - Department of Biomedical Sciences, Creighton University, Omaha, NE.
Aminoglycosides are a class of antibiotics frequently prescribed to treat ear infections because of their effectiveness and relative low cost. Ototoxicity and permanent hearing loss are recorded in 20-50% of patients treated with aminoglycosides for bacterial ear infections. Ototoxicity due to aminoglycosides is associated with inflammatory responses in the cochlea. Long noncoding RNAs (lncRNA) can act as cell-specific regulators of inflammatory gene transcription. We are investigating how inflammatory signaling followed by aminoglycoside exposure influences lncRNA expression, inflammation, and ototoxicity. In vivo animal model systems mimicking bacterial infection and aminoglycoside-induced hearing loss are being used to evaluate the expression of lncRNAs in the central nervous system and cochlea. Preliminary data indicates that linc-Tnfaip3 and lincRNA-Cox2 are upregulated following inflammatory responses induced by intraperitoneal injection of the bacterial antigen lipopolysaccharide (LPS) in mice. We hypothesize that differentially expressed lncRNAs like lincRNA Tnfaip3 and Cox2 upregulate proinflammatory gene expression and underlie aminoglycoside induced ototoxicity. RT-qPCR analyses of CNS tissue demonstrates the upregulation of these lincRNAs in. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were used as the LPS- induced proinflammatory mouse model system. In situ analyses are underway to investigate cellular localization of linc-Cox2 and linc-Tnfaip3 expression. Single cell sequencing analyses of cochlear cells demonstrated differential regulation of proinflammatory genes. RT-qPCR analysis confirms differential gene expression. A more robust understanding of the mechanisms of inflammation in the cochlea and the mechanisms of aminoglycoside-induced ototoxicity could provide therapeutic targets for the treatment or prevention of hair cell death and hearing loss following antibiotic delivery for ear infections. This publication was made possible by grants from the National Institute for AIDS and Infectious Disease (NIAID) (1 R15 AI156879) and the National Institute for General Medical Science (NIGMS) (5P20GM103427), components of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS or NIH.
DEVELOPMENT OF VAPING PEANUT ALLERGY MOUSE MODELS
Marissa Hoover1, Joseph Roeder1, Zane Carlson1, and Joseph J. Dolence, Ph.D.1
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
While vaping has become popular in recent years, the health effects of vaping remain unclear, especially how it impacts the immune responses that originate in the lung. We have shown that peanut (PN) allergy can be induced via inhalation in mice and recent data suggests this likely happens in humans. In this study, we developed mouse models to ask whether vaping can influence the ability of the immune system to mount allergic responses against PN. First, we showed that mice sensitized using PN solution containing vape juice displayed decreased PN-specific IgE responses and milder anaphylaxis. To further study, we sensitized mice using electronic conditioned media (ECM) to expose mice to the vapor. To make ECM, we bubble vapor into the media used to expose the mice to PN and ask whether the vapor itself alters allergic responses. Preliminary data suggested mice exposed to 3 mg/mL nicotine ECM displayed lower PN-specific IgE. Currently, we are using 6 mg/mL nicotine ECM to examine whether this drives greater suppression. We next plan to examine whether ECM influences the ability of B and T cells to respond to PN using flow cytometry. Taken together, this data suggests that vaping stifles the ability of the immune system to mount immune responses to the clinically relevant PN antigen.
EXAMINING HOW SEX DIFFERENCES INFLUENCE THE ABILITY OF NEUTROPHILS AND DENDRITIC CELLS TO MOUNT RESPONSE FOLLOWING INHALATION OF PEANUT
Dana Dubas1, Tyler Shaner1, Sunanda Rajput1, McKenna S. Vininski1, Nicholas J. Hobbs, Ph.D.1, and Joseph J. Dolence, Ph.D.1, dubasd@lopers.unk.edu,
1 - Department of Biology, University of Nebraska at Kearney, Kearney, NE.
Knowledge of how innate immune cells respond to peanut (PN) to elicit PN allergy remains incomplete, especially how sex differences impact the ability of these cells to respond to PN. This study compared male, female, and androgen receptor-deficient (ARTfm) male mice exposed to PN via inhalation in 3-day mouse models to elucidate how sex differences impacted the response of neutrophils and dendritic cells (DCs) to PN. We found that DCs, but not neutrophils, responded following PN inhalation. We further studied DCs by examining CD103+ CD11b- cDC1s and CD11b+ CD103- cDC2s, two DCs shown to capture PN following inhalation in mice. cDC1s and cDC2s were found to express IL-1R1 in response to PN. IL-1R1+ cDC1s responded to PN in WT male and female, but not in Tfm male mice. IL-1R1+ cDC2s reacted to PN in WT male and female, along with displaying a more abundant response in Tfm male mice. We are currently expanding this study to examine whether these DC populations are impacted following 11 days of PN inhalation to investigate how sex differences impact the response of DCs at a time point just before PN-specific adaptive responses begin to elicit PN-specific IgE. Collectively, this data suggests that IL-1R1+ cDCs play an important role in response to PN, likely activated by IL-1 alpha released by lung epithelial cells after PN inhalation. The differential response of cDCs in the Tfm mice suggest that male sex hormones, namely androgens, play a role in regulating the ability of DCs to mount initial response to PN.
AFTERNOON SESSION B4
ANALYSIS OF THE MICROBIOME OF ORNATE BOX TURTLES
Blake Skoumal¹, Victoria Martinez², Franziska C. Sandmeier², Erin Doyle¹
1- Department of Biology, Doane University, Crete, NE;
2- Department of Biology, Colorado State University- Pueblo, Pueblo, CO.
The ornate box turtle (Terrapene ornata) is a species of North American turtle known for its distinctive and intricate shell patterns, which vary among individuals. Found ingrasslands, prairies, and open woodlands of the central United States, these terrestrial turtles display a remarkable ability to withstand temperature extremes. Ornate box turtles exhibit omnivorous feeding habits, consuming a diet that includes insects, vegetation, and fruits. Their population faces threats from habitat loss and fragmentation, making conservation efforts crucial for the preservation of this visually striking and ecologically significant species. Due to their asocial and isolated lifestyle as well as the lack of non-gastrointestinal microbiome studies of reptiles, we are very curious where their microbiomes’ originate. The microbiome of an organism is classified as the assortment of microorganisms found on or within an organism’s cells and/or biofluid. A healthy reptilian gut microbiome consists mainly of proteobacteria and occasionally an abundance of bifidobacteria depending on diet. We will use metagenomic data collected in order to draw comparisons between each sampling location. Full metagenomic shotgun sequencing will be performed as well as analysis of 16S ribosomal subunit sequencing of samples collected in the turtle’s burrow, inside the turtles’ nasal cavities and the air surrounding the turtles’ habitat, all in order to determine where different components of the turtles’ microbiomes are acquired from and how they are transmitted to the animals themselves.
MEASUREMENT OF BINDING CONSTANTS FOR FASCIOLA HEPATICA FATTY ACID BINDING PROTEIN (FH-V) AND VARIOUS HYDROPHOBIC LIGANDS
Frank A. Kovacs1 and Mahesh Pattabiraman1, kovacsfa@unk.edu
1 - Department of Chemistry, University of Nebraska-Kearney, Kearney, NE.
Fatty acid binding proteins (FABPs) are a family of small structurally conserved proteins that bind reversibly to hydrophobic ligands inside of cells and chaperone them to various locations including the nucleus and mitochondria. Fasciola hepatica (Fh), is a parasite that causes a foodborne infection that spreads between animals and humans. One of its FABPs, Fh-V has been implicated as playing a role in drug resistance of the organism to TCBZ, the most effective drug known to combat the parasitical infection. This study presents our initial results for the expression, purification and characterization of Fh-V and its binding to TCBZ-SO and some hydrophobic ligands using a fluorescence displacement assay and isothermal titration calorimetry.
ACCESSING GERM TUBE FORMATION AND FARNESOL RESPONSE IN CANDIDA ALBICANS
Shyanne Urbin1, Daniel J. Gutzmann1, Kenneth Nickerson1, and Audrey Atkin1, surbin2@huskers.unl.edu
1 - Department of Biological Sciences, University of Nebraska - Lincoln, Lincoln, NE.
Candida albicans is a commensal fungus that inhabits the throat and gastrointestinal tract of a large proportion of humans. Typically, Candida albicans causes no issues in a healthy individual, but in those that are immunocompromised, it can be responsible for an opportunistic infection. One link between Candida albicans and this virulence is its ability to morphologically switch between yeast and hyphal cells. This process of switching from yeast to hyphal cells is mediated by the formation of germ tubes, which involves several regulatory pathways. One such way where germ tube formation is inhibited is by a molecule farnesol, which Candida albicans produces as a quorum sensing molecule. Since Candida albicans can both produce and respond to farnesol, it is important to elucidate the mechanisms between these systems. In this presentation, this connection is investigated. To access the systems that are involved in farnesol response, a germ tube assay was conducted with a collection of knockout mutants that were previously screened for farnesol production. From this study, swi4Δ/Δ and nrg1Δ/Δ were identified as farnesol non-responsive. In addition, ahr1Δ/Δ and msn4Δ/Δ were considerably not as responsive at lower farnesol concentrations than the wildtype. This suggestion of swi4 and nrg1 being important for responding to extracellular farnesol within Candida albicans will be discussed in this presentation. Since swi4 is implicated as a downstream effector of the cell wall integrity pathway in Candida albicans, the hypothesis that this indicated will be elaborated upon. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #5P20GM103427.
CHARACTERIZATION OF GUSTATORY RECEPTORS FROM WESTERN CORN ROOTWORM
Hope Hixson1 and Dr. Hideaki Moriyama2, hhixson3@huskers.unl.edu
1 - Department of Biochemistry, University of Nebraska, Lincoln, NE;
2 - School of Biological Sciences, University of Nebraska, Lincoln, NE.
Organisms have chemical sensors with high sensitivity and wide dynamic range. Our long-term goal is to understand these structures and functions, and scrutinize them as sensors. We began our research with the simpler system of insects. Insects use carbon dioxide gas as a way to sense the location of interested objects, such as humans and plants. Insects like mosquitoes and fruit flies sense carbon dioxide gas through gustatory receptors (GR) on the upper pedipalps. There are three major homologous gustatory receptors identified in insects, GR1, GR2, and GR3, however, it is yet to be determined whether these receptors respond to carbon dioxide or bicarbonate. We chose GR genes from western corn rootworm (Diabrotica virgifera virgifera) as a model. Our beginning aim was to reconstitute functional carbon dioxide receptors in Xenopus laevis oocytes. Following this, we would like to clarify the division of functions between GR2 and GR3. To understand the chemical signal that stimulates these receptors we synthesized genes corresponding to GR1-3 plus a pseudogene, cloned each of them separately into pNCB1 vectors, and produced mRNA for each. Next, we expressed them in Xenopus oocytes and subjected them to a two-electrode voltage clamp (TEVC) as a function of CO2 concentration. Our current aims are to differentiate the roles of GR2 and GR3 and determine how the concentration of sodium bicarbonate solution effects induced current response.
AFTERNOON SESSION-C4
KINETIC STUDIES OF PROTEINS INVOLVED IN GENE SILENCING USING SURFACE PLASMON RESONANCE
Ivy Williams1 and Lynne Dieckman1, IvyWilliams@creighton.edu
1 - Department of Chemistry and Biochemistry, Creighton University, Omaha, NE.
Genomic DNA in eukaryotes must be tightly packed to fit inside the cell nucleus. To properly organize DNA, it is coiled around eight histone proteins to form nucleosomes, which are further condensed to form chromatin. Depending how tightly the nucleosomes are packed dictates whether a gene will be expressed or silenced. The regulation of nucleosome assembly and gene silencing requires the coordinated action of two key proteins: proliferating cell nuclear antigen (PCNA) and chromatin assembly factor-1 (CAF-1). PCNA is a homotrimeric sliding clamp protein that encircles the DNA double helix and recruits CAF-1 to the replication fork to deposit histone proteins on newly synthesized DNA. If PCNA and CAF-1 are unable to interact, gene silencing cannot occur, resulting in genome instability and disease. The precise mechanism governing the PCNA-CAF-1 interaction still remains unclear. The goal of my research is to determine the kinetics of binding between PCNA and CAF-1 using surface plasmon resonance (SPR). I have determined that the PCNA-CAF-1 interaction occurs with a 2-step mechanism of binding and an affinity comparable to other PCNA binding partner interactions. Additionally, our lab has identified two different PCNA binding motifs within CAF-1, and the kinetics of binding of these CAF-1 motifs to PCNA will be quantified using SPR in the future. Coupling my kinetic data with other structural and thermodynamics studies of the PCNA-CAF-1 interaction obtained in the lab will provide a comprehensive understanding of how CAF-1 and PCNA interact, and thereby how these proteins regulate nucleosome assembly to maintain genomic stability.
STRUCTURAL ANALYSIS OF CRASSOSTREA GIGAS OAZ-PK RNA
Hannah Ladwig1, Rhiannon McCracken1, and Juliane Soukup1, hml53613@creighton.edu
1- Department of Chemistry and Biochemistry, Creighton University, Omaha, NE.
Riboswitches are a type of non-coding RNA that regulate downstream gene expression upon metabolite binding. When a riboswitch interacts with its ligand, it undergoes a conformational change resulting in a change in gene expression. This change in gene expression operates as a feedback mechanism, affecting the same metabolic pathway in which the ligand functions. The Soukup Lab researches potential eukaryotic riboswitches within the Ornithine Decarboxylase Antizyme pseudoknot (OAZ-PK) RNA. One such potential riboswitch is found in the OAZ-PK RNA of a species of oyster, Crassostrea gigas, which is believed to interact with various natural and non-natural polyamines. In-Line Probing (ILP) experiments can be used to analyze the structural changes of this RNA segment upon binding to differing concentrations of these polyamines. Preliminary data from ILP experiments with Crassostrea gigas OAZ-PK RNA indicate that the binding of spermine results in a structural change to the RNA segment, but not other polyamines. Current experiments aim to examine the presence of a structural change upon binding of a closely related polyamine, spermidine. Riboswitches have demonstrated significant impacts on the regulation of metabolic pathways in bacteria, and thus are being used as a target of possible antibiotic treatments. Identification of similar riboswitches in eukaryotic species will provide an opportunity for the development of novel anti biological agents.
The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
INVESTIGATING THE FUNCTION OF THE TRANSCRIPTION ELONGATION FACTOR, GREA, IN CHLAMYDIA TRACHOMATIS GROWTH AND DEVELOPMENT.
Abigail Swoboda1,2, Scot Ouellette1, abigailswoboda@unomaha.edu
1 - Department of Pathology, Microbiology, and Immunology, University of Nebraska Medical Center, Omaha, NE;
2 - Department of Biology, University of Nebraska Omaha, Omaha, NE.
The obligate intracellular pathogen, Chlamydia trachomatis (Ctr), undergoes a complex developmental cycle where the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, and non-dividing form that initiates infection of a host cell whereas the RB is the larger and non-infectious form that replicates within a membrane-bound vesicle termed an inclusion. The mechanism(s) underlying transcriptional changes in the developmental forms remains poorly understood but is important for understanding how Ctr transitions between EBs and RBs. GreA is a transcription elongation factor, which executes anti-arrest activity to restore RNA polymerase transcription elongation. Although chlamydial GreA is homologous to canonical GreAs, it possesses an unusually extended N-terminus region of unknown function. We hypothesize that the Ctr greA gene is expressed as two distinct products that may contribute to developmental progression. To test this, we investigated the impact on Ctr growth and development of (i) expressing different isoforms of the greA gene and (ii) altering GreA expression. By assessing bacterial morphology and infectious progeny production, we found that overexpression of the GreA N-terminus or CRISPRi-mediated knockdown of greA elicited a negative impact on Ctr development. The growth defect during knockdown was partially complemented by expression of full-length GreA. We also observed evidence for an internal promoter that drives expression of the C-terminus alone, suggesting that the greA gene may be transcribed as two separate gene products. These data implicate GreA as essential for Ctr growth and development.
USING BACTERIOPHAGES TO TREAT PLANT INFECTIONS OF P. SYRINGAE
Sierra Laschanzky1, Katelyn Jindra1, Erin Doyle1, sierra.laschanzky@doane.edu
1 - Department of Biology, Doane University, Crete NE.
Bacteriophages are viruses that infect bacteria. Bacteriophages have the potential to be used in a process called phage therapy, where a bacterial infection is treated with these viruses instead of/alongside antibiotics or other treatment methods. Phage therapy in plants has been tested before, and has shown promising results. However, more research is needed before this treatment option becomes a truly viable method. We isolated and characterized 14 bacteriophages from soil samples collected across Nebraska using the host bacteria Pseudomonas syringae pv. tomato DC3000. This bacteria strain is specific to tomato plants. Plaque count and size were found to be different between wild type Pst and lab mutant Pst, and restriction enzymes showed that all 14 isolated phages are genetically distinct. My research goal will be to characterize each phages’ attachment rates and burst sizes, so that I can choose the best suited phages for my plants’ phage therapy. Once chosen, we will make a cocktail of the chosen phages and test their viability in curing Pst infections in planta. We will also compare the efficacy of the individual chosen phages vs the cocktail.
ENAMEL EROSION DUE TO ENERGY DRINKS
Jaylee Shaw1 and Dr. Mary Keithly1, jaylee.shaw@eagles.csc.edu,
1 - Department of physical sciences, Chadron State College, Chadron, NE.
Energy drink consumption is an increasing trend throughout our society. A side effect of these drinks is the erosion they can cause to tooth enamel. Enamel erosion occurs through demineralization of the tooth, which energy drinks can cause due to their acidic properties. Examination of the damage caused to the enamel of teeth from energy drink consumption was completed. The effects of rinsing with water after exposure versus not rinsing, along with whether caffeine or sugar causes more damage were analyzed. Prior studies of this phenomenon served as a basis for analysis of new brands of energy drinks. Little research on Bubbl’r and Celsius energy drinks exists as they are fairly new to the market. Before beginning, the mass and amount of calcium, phosphate, and elements smaller than magnesium were measured in each bovine tooth using portable X-Ray fluorescence. The pH of each drink was also recorded. The teeth were submerged in the designated drinks for two hours per day. Drinks were carbonated every time to ensure consistency between trials. In between testing, the teeth were stored in synthetic saliva. Analysis of data is currently in progress and expected to show which energy drink causes the most damage to tooth enamel.
AFTERNOON SESSION-D4
TOWARDS A MATHEMATICAL UNDERSTANDING OF PRION STRAIN DYNAMICS
Josh Gilbert1, Clay Cressler1, and Jason C. Bartz2, jgilbert19@unl.edu
1 - School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE;
2 - Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE.
Prion diseases are caused by the template-driven misfolding of a host-encoded protein to an aberrant isoform that can cause the conformational conversion of susceptible monomers to the disease-associated conformation. These diseases are always fatal and are unique in that they can have a sporadic, hereditary, or infectious etiology. Even though the agent lacks a known informational nucleic acid, distinct and heritable phenotypes of disease are observed. These differences are referred to as strains and their variable information is thought to be encoded by the conformation of the disease-associated protein. Prion strains have been shown to act in a dependent manner when coinfecting the same populations of cells. Specifically, two strains can interfere with one another. This is thought to mostly be due to competition for susceptible monomers. Interestingly, prions are capable of adapting to novel selective pressures very quickly. This has led to the hypothesis that prions are composed of a spectrum of distinct conformations. We now extend mathematical models that help explain prion conversion kinetics to the idea that prion strains compete with one another for susceptible monomers. This is the first step towards bridging the idea of a conformational spectrum to the interactions between different prion conformations within that spectrum.
SELECTIVE INTERACTIONS OF MAMMALIAN PRION PROTEINS TO THE SURFACE OF LIPID MEMBRANES: A COARSE-GRAINED MOLECULAR DYNAMICS SIMULATIONS STUDY
Noah Greenwood 1 , Jamie Janos 2, Mason Borgman 2, Patricia Soto1, NoahGreenwood@creighton.edu
1 - Department of Physics, Creighton University, Omaha, NE;
2 - Department of Chemistry, Creighton University, Omaha, NE.
The Prion protein is the molecular hallmark of the incurable prion diseases affecting mammals, including humans. Notable instances include Creutzfeldt Jakob Disease in humans and Chronic Wasting Disease (CWD) in cervids. The protein-only hypothesis states that the misfolding, accumulation, and deposition of the Prion protein play a critical role in toxicity. Mammalian prion proteins, including Doppel and PrPC, despite their similar globular domain topology and lipid raft anchoring, selectively misfold only in the case of the prion protein, PrPC, contributing to the pathology of prion diseases. To develop a structural model of Prion protein conformational conversion, it's crucial to understand the underlying biophysical forces governing Prion protein-lipid interactions in physiological conditions. Our study delves into elucidating the interactions of globular PrPC and Doppel protein with the membrane surface and their distinct responses. Through coarse-grained molecular dynamics simulations, we explore the influence of protein sequences, topologies, and GPI anchoring on protein-membrane associations. Our simulations reveal that the interplay between proteins and lipids, as well as the conformational constraints imposed by GPI anchoring, collectively promote preferential binding of PrPC via the loops adjacent to α-helix 2, however these interactions are absent in Doppel protein simulations. We observe that polar residue side chains within the loops of PrPC, possessing specific functional groups, interact with the membrane, leading to varying degrees of insertion. The implications of these observed binding modes on the misfolding and aggregation of PrPC will be discussed, along with insights into potential protective mechanisms against misfolding. We anticipate that experimental studies utilizing techniques such as atomic force microscopy and spectroscopy will further probe membrane remodeling induced by the Prion protein.
INVESTIGATING ANXIOLYTIC RESPONSES OF DANIO RERIO THROUGH THE ROLE OF N-ACETYLCYSTEINE AMIDE ON THE GLUTAMATERGIC NEUROTRANSMITTER PATHWAY
Brooklynn K. Schmidt1 and Ryan Wong1.2
1 - Department of Psychology, University of Nebraska at Omaha, Omaha, NE;
2 - Department of Biology, University of Nebraska at Omaha, Omaha, NE.
Anxiety disorders are considered a common mental health issue across the world. These disorders can manifest when there is a dysregulation of neurotransmission in the central nervous system. Studies have linked increased glutamate levels as a result of stress, and such unregulated levels have led to a variety of neurodegenerative disorders, weakened synapses, and reduced synaptic plasticity. N-acetylcysteine amide (NACA) reduces the presynaptic release of glutamate, regulating glutamate transmission and minimizing the effects of anxiety. Proactive and reactive stress coping styles are two ways animals respond to stress and anxiety. Proactive individuals tend to show lower stress and anxiety behaviors as they are more risk-prone, while reactive individuals tend to show the opposite as they are risk-averse. The role of glutamate in facilitating the display of alternative stress coping styles is not well understood. We will test the hypothesis that treatments of NACA will inhibit stress and anxiety by decreasing glutamate neurotransmission via mGluR2/3. After acute administration of NACA to the animals, we will measure behavioral changes in stress and anxiety (light/dark test) followed by immunohistochemistry to quantify active mGluR2/3 neurons. Analyzing NACA’s effects will contribute to future advancements in drug treatment therapies by providing a better understanding of glutamate neurotransmission between the different stress coping styles and giving insight into how the mechanism of glutamate modulation may alter anxiety. The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427.
3D TISSUE MORPHOMETRY FOR NPRT AGAINST BRAIN TUMORS
Joshua Taylor1, Dylan Bui1, Jordyn Rockwell1, and Andrew Ekpenyong1, jot07134@creighton.edu
1- Department of Physics, Creighton University, Creighton, NE.
This work joins the decades-old search for appropriate nanoparticles that mediate the delivery of highly localized and enhanced doses of radiation to tumors that are radioresistant such as glioblastomas. Nanoparticle mediated radiotherapy (NPRT) is a multidisciplinary field, and our lab uses NPRT to target glioblastoma, one of the most aggressive forms of brain cancer with dismal survival rates. Here, we study the efficacy of NPRT against glioblastomas, using fluorescence-guided morphometry. We have also gone from 2D cell layers to 3D tissue spheroids. We have maintained the same dyes used in the 2D assays: Hoechst and Calcein. The in vitro radiotherapy is carried out using a standard cell irradiator (Faxitron). Following fluorescence imaging of the spheroids, we quantitatively and qualitatively analyze the images using ImageJ’s fractal analysis plugin, FracLac, to extract parameters such as lacunarity. Our preliminary results for lacunarity at early time points (24 to 48 hours) will consolidate our later time clonogenic assays (14 to 21 days) as we decipher treatment conditions that promise better therapeutic outcomes.
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