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CORAS Poster Presentation Themes and Abstracts Celebration of Research and Scholarship Wednesday, February 10, 2021 POSTER PRESENTATION THEMES AND ABSTRACTS Basic Science and Oral Biology Poster#1 New Directions in Understanding Arrabidaea chica Modulation of Inflammatory Pathways Walton Godwin, Carolina Maia, Silvana Pasetto, Ilza Maria de Oliveira Souza, Mary Ann Foglio, Ramiro Murata. Objectives: Oral mucositis is the painful inflammation and ulceration of oral tissues as a side- effect of aggressive cancer therapies. Activation of the innate immune response by DAMPs/PAMPs are important components in the initiation and severity of mucositis. Currently, there is no treatment or cure for mucositis. Arrabidaea chica has been traditionally used as a wound healing agent for its anti-inflammatory properties. In this study, the anti-inflammatory effects and underlying cell signaling transduction of A. chica extract in LPS/Zymosan-stimulated oral fibroblasts and THP-1 cells was investigated. Methods: Human gingival fibroblasts (HGF-1) and monocytes (THP-1) were cultured. The cytotoxicity of A. chica extract was determined by 24- hour exposure of cells at concentrations from 0.025-250μg/mL. Cell viability assays were then performed to determine the LD50. HGF-1 and THP-1 were exposed to stimulating concentrations of LPS and/or Zymosan with or without A. chica extract. Sample supernatants were collected from samples at 0, 3, and 6-hour time points. Cytokine production was determined by Luminex analysis of the supernatants. Inflammatory pathway modulation was/will be investigated using Simple Western blot techniques and RT-PCR. Results: Viability assays determined that A. chica extract is not toxic to HGF cells in low concentrations. Luminex assays indicated that cells exposed to A. chica extract expressed lower amounts of pro-inflammatory cytokines IL-6 and IL-8 when exposed to LPS than cells without extract. Cells exposed to A. chica extract also expressed higher amounts of anti-inflammatory cytokine IL-10. Conclusions: Arrabidaea chica extract inhibits pro- inflammatory cytokine expression and promotes anti-inflammatory cytokine expression in human gingival fibroblasts after exposure to LPS. This study is now implementing RT-PCR, simple western blot, and Luminex assay technologies to investigate the signal transduction proteome, transcriptome, and expression of multiple inflammatory cytokines in monocyte cells. New methods of studying this pathway are also now being implemented, including DNA and RNA sequencing. Poster#2 Saliva Sampling as a Source for SARS-COVID 19 RT-qPCR Detection Silvana Pasetto, Ming Fan, Mark E. Moss, Suzanne Lea, Ramiro M. Murata, Rachel Roper. Background: The diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection relies on the detection of viral RNA by RT-qPCR conducted with respiratory specimens such as nasopharyngeal swabs. However, this procedure requires specialized personnel, centralized laboratory facilities, and time to provide the results. Saliva has been increasingly used over the last few decades for evaluating human health. In recent days, a test for assessing the RNA in saliva samples was approved by the US Food and Drug Administration. The use of saliva as a diagnostic specimen has advantages such as it is easily self- collected by the patient with no discomfort, and it reduces the risks for the operator. Objective: The objective of this project is to substantiate the use of saliva sampling as a noninvasive analysis of Covid- 19 infection. Methods: 7 SARS-RNA 5x10 /equivalent (1:1000, 1:10,000 and 1:100,000), saliva-5μL (only), saliva plus SARS- CoV-2- RNA were mixed with oligonucleotides primers detecting nucleocapsid gene (N1 or N2), and saliva plus SARS-RNA RNase P primers to detect human RNase P (to show that adequate sample was collected for control) were mixed with Reaction Master Mix for detection of COVID- 2019 virus and amplified by RT-PCR equipment (QuantStudio 3 – Thermo Fisher). Results and Conclusions: The initial experiments provided positive results for the 3 SARS-RNA dilutions and Saliva plus SARS-RNA. The highest dilution (1:100,000) showed 2.75 genome copies could be detected with both primers N1 and N2. Saliva plus primers for RNaseP gave a positive result, and saliva only plus SARS-CoV primers were negative. The Ct values were between 21 to 33 (≤40) as requested by CDC for detection of COVID-19). With this data it was concluded that the role of salivary diagnostics is promising for direct testing for SARS- Covid19 as well as being easy, fast, and inexpensive. Additionally, the sensitivity of the salivary sample is comparable to that of respiratory samples. Poster #3 Understanding the Role of Rab10 in Neuronal Resilience Wyatt Bunner, Denys Bashtovyy, Rachel Dodson, Olivia Thiery, Vishwanath Vasudev Prabhu, Tuan Tran Erzsebet Maria Szatmari. Objectives: Advanced age and presence of ApoE epsilon allele are major risk factors for Alzheimer’s disease (AD). However, a small percentage of elderly and carriers of ApoE epsilon, do not develop AD. The molecular basis of resilience to AD is not fully understood. A loss of function mutation in the Rab10 gene was shown to confer a 40% reduction in AD risk, even in patients homozygous for ApoE epsilon allele. Here we describe our results on cellular, molecular, and behavioral characterization of Rab10+/- mice, that we created to study the cellular and molecular mechanisms of Rab10-dependent neuronal resilience in a mouse model of AD on Rab10+/- background. Methods: Behavioral studies: Open field, Object in place (OIP), Morris water maze, novel object recognition task. Biochemistry: Western blotting to evaluate Rab10 reduction in the brain of Rab10+/- mice. Immunofluorescence: Coronal brain sections stained with anti-NeuN antibody to evaluate the effect of Rab 10+/- on brain morphology. Transcriptome profiling: Gene expression analysis using the nCounter neuropathology panel. Results: The level of Rab10 in the brain of Rab10+/- mice was reduced by approx. 50% compared to their Rab10+/+ littermates. Interestingly, we noticed significant difference in body weight between genotypes, that was sex-dependent: female Rab10+/- mice’s body weight compared to their Rab10+/+ littermates. Next, we performed a battery of behavioral testing. Our results show that Rab10+/- mice perform significantly better in OIP task that tests hippocampus-dependent spatial memory. To elucidate the possible molecular basis of enhanced memory formation, nCounter profiling of the cerebral cortex was performed. Rab10+/- mice had a significant change in the expression of multiple genes associated with neuronal survival (Synaptotagmin-4 and Syntaxin 2). Conclusion: Rab10 functions as a negative regulator of hippocampal learning and memory formation. Understanding the molecular mechanisms of neuronal resilience to disease may hold important clues for the design of novel neuroprotective strategies. Poster #4 Engineering New Bioactuators for the Optical Manipulation of Dendritic Spine Robert M. Hughes, Anna Werz, Erzsebet Szatmari, Wyatt Bunner. One critical, yet underexplored area in dendritic spines dynamics is that of the proteins responsible for the biomechanical initiation of dendritic spine formation. One family of proteins that has been previously characterized as responsible for inducing membrane curvature and subsequent spine formation are the BAR (Bin, Amphiphysin and Rvs)-domain containing proteins. BAR domains are recruited to the interior plasma membrane in response to phosphoinositide signaling, where they induce mechanical stress on the membrane. BAR and F-BAR domains, for example, induce positive membrane curvature and subsequent invagination, while I-BAR domains induce negative membrane curvature and subsequent protrusion. In this proposal we will couple I-BAR domains with an optogenetic switch (Cry2/CIB) to develop a novel optogenetic tool (Cry-BAR) for the induction of spinogenesis (dendritic spine formation), a critical step in synaptogenesis (generation of new synapses) and communication between neurons in the mammalian brain. This tool will be a first-in-class modulator of the biomechanical forces responsible for the plasma membrane protrusions that ultimately mature into dendritic spines. Cry-BAR will be tested and validated in neuron cultures from newborn mice using techniques and instrumentation previously developed as a result of collaborative work from our team. Poster #5 Role of ADAP-1/ Centaurin α1 In AlZheimer’s Disease Erzsebet Maria Szatmari, Wyatt Bunner, Denys Bashtovyy, Rachel Dodson, Olivia Thiery. Objectives: Synaptic failure in Alzheimer’s disease (AD) is caused by accumulation of the toxic peptide beta-amyloid (Aβ42). Previous studies from our lab found that the brain-specific Ras- anchoring protein, ADAP-1/Centaurin α1 (CentA1) is involved in Aβ42-induced neuronal dysfunction. To evaluate the role of CentA1 in vivo, we created CentA1 knockout mice (CentA1 KO). To test the role of CentA1 in AD pathomechanism, we crossed the CentA1 KO mice with the J20-hAPP mouse model of AD (J20xCentA1 KO). Methods: Immunofluorescence: NeuN staining of coronal brain sections to evaluate brain morphology Biochemistry: Western blotting to validate CentA1 KO and APP overexpression in transgenic mice Behavioral studies: Morris water maze (MWM) test to evaluate spatial memory Golgi staining and spine density analysis in the SLM region of the hippocampus Immunohistochemistry staining for plaques (6E10) and neuroinflammation (Iba-1 and GFAP) Transcriptome profiling using
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