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It is with great pleasure that we welcome you to the first MBIM Undergraduate Research Symposium! The creation of this forum was inspired by the outstanding research being done by students in the microbiology and immunology undergraduate program at the University of British Columbia. The MBIM program provides several opportunities for students to gain research experience. As you peruse the abstracts and take in the presentations, it might be helpful to consider the types of courses that support the work presented here:

Directed Studies courses (MICB 448 and MICB 449) provide students with an opportunity to do research in academic research laboratories in our department.

Course-based Undergraduate Research Experiences (CUREs) (MICB 447 and MICB 421) provide students with a team-based opportunity to lead their own research project. Teams develop a research question, execute experiments in the laboratory, and communicate their findings in the Journal of Experimental Microbiology and Immunology.

In ‘Topics in Molecular Virology’ (MICB 406) students work through a theoretical research project with the goal of generating a short educational scientific article that addressed “hot” topics (PEARLS) of relevance and importance within the field of molecular virology. The best educational scientific articles written by the MIBC406 students are published in JEMI PEARLS.

Co-op experiences often take students into academic research labs where they engage in a broad range of projects.

With 42 abstracts submitted in its inaugural year, you have given the MBIM Undergraduate Research Symposium an excellent start! Your abstracts have been organized into four groups encompassing topics in bacteriology, immunology, new concepts in virology, and the biology of infection across diverse viral systems. For a bit of fun each group has been assigned a classic rock theme song – check out yours sometime if you haven’t heard it before! Contributing authors are cited and the presenting authors are denoted with an asterisk (*). The courses from which each study derived are also listed. The symposium has two sessions of oral talks (A and B). Each session has two rooms (Wesbrook 100 and MSL 100), each with 6 talks. The sessions are titled A-WESB, A-MSL, B-WESB, and B-MSL. Shivani Mysuria and Sunny Chen have selected a blend of 6 talks for each session that will integrate directed studies/co-op research, CURE projects, and PEARLS presentations. And of course, sandwiched between the talks is the poster/networking session. We are proud of our students. Throughout the year we observe dedicated effort supported by scientific rigour and research integrity. This symposium brings together many of the students from this exceptional undergraduate community. The presentation of research findings, the sharing of new ideas, and formation of connections is a fundamental part of doing science. We commend you for seizing this opportunity to advance your scientific experience and education.

We hope you enjoy the symposium and come back to support our undergraduate research program in future years!

Keep on rocking! Drs. David Oliver and François Jean, Co-Chairs, 2018 MBIM Undergraduate Research Symposium

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A Message from the Head The goal of our M&I undergraduate program is to develop citizen-scientists who are knowledgeable and knowledge-seeking, and who have the skills to be effective, self-directed lifelong learners. Participating in curiosity-based research is one of best ways to acquire those skills. Your enthusiasm for the different types of research and learning experiences that our department offers has been terrific. I’m looking forward to seeing all the great work that you have done and to hearing about your experiences in M&I. See you on April 26!

Mike Gold Professor and Head Department of Microbiology and Immunology University of British Columbia

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Many thanks to the organizing committee members from the Department of Microbiology and Immunology.

David Oliver, PhD (Co-chair) - Instructor François Jean, PhD (Co-chair) – Associate Professor Parvin Bolourani, PhD - outreach, alumni engagement, postdoctoral fellows & project coordinator Sunny Chen - undergraduate student Kim Dill-McFarland, PhD – post-doctoral fellow Mike Gold, PhD – Department Head Craig Kornak – undergraduate administrative assistant Shivani Mysuria – undergraduate student Andrew Sharon, BSc – graduate student

Special thanks to Eric Lee and Patrick Ho for IT support and web development. John Nomellini is gratefully acknowledged for help with logistics. Support for this symposium has been generously provided by the UBC Department of Microbiology and Immunology and ECOSCOPE.

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Symposium Program – Thursday 26th April 2018

Registration 12:00 - 12:15 pm Wesbrook Lobby Welcome and Keynote Lecture 12:15 - 1:20 pm Wesbrook 100 Session A – Oral Presentations 1:30 – 2:45 pm WESB 100

MSL 100 Poster Session 2:45 – 4:00 pm MSL 101 Session B – Oral Presentations 4:00 – 5:15pm WESB 100

MSL 100 Closing Remarks and Awards 5:15 - 5:30pm MSL 100

WESB – Wesbrook Building MSL – Michael Smith Building Oral Talks:

Presenters are asked to check in with the convener before your session begins.

Talks are limited to no more than 8 minutes. A 2 minute question period will follow.

Posters:

Presenters are asked to have at least one author stand by their poster during the assigned poster session.

Posters can be picked up from Craig. Please put your poster up on your assigned board in MSL101 before 12pm

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Oral Talks Session A-WESB (1:30-2:45pm)

1. Uyen Nguyen

2. Maria-Elizabeth Baeva

3. Felix Ma

4. Wumian Wang, Myra Lam

5. Amanda E Krystal*, Reiko Okamoto*, Andrea Sze*, Zachary F Weiss*

6. Kate W. Huang*, Erika L. Jang, Emily E. Kamma*, Annie H. Wu

Session A-MSL (1:30-2:45pm)

1. Ian Lee, Felix Ma, Daniel Chan, Abigail Morres

2. Thomas Hoang

3. Leo T Liu

4. Jordan Brundrett

5. Priya Suresh

6. Jake Johnston*, Pierre Becquart, Jacqueline Quandt

Session B-WESB (4:00-5:15pm)

1. Karl Angelo Abuan*, Haya Abuzuluf*, Yuxin Ban*, Leilynaz Malekafzali*

2. Tamar V. Av-Shalom

3. Andrew P. Golin

4. Niknaz Malekafzali

5. Syed S Raza

6. Mitch Syberg-Olsen

Session B-MSL (4:00-5:15pm)

1. Laurie Chang*, Jessie Zhang*, Jenna Ching, Regina Onal

2. Chris James, Ofodile Morah, Victoria Panwala, Amin Yarmand*

3. Boyuan Zheng

4. Sami Rahman

5. Shivani Mysuria

6. HT Le, J Atif*, DL Mara, B Castellana, J Treissman, AG Beristain.

Presenting authors are denoted with an asterisk (*) following their name.

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Posters Codes / Authors

1P Michelle Gu, Almas Khan, Desiree S. Pagulayan, Karen Tam 2P Jordan Brundrett, Jumar Orach, Lukas Sonderegger, Anthony Yan 3P Vivian CW Fung*, Yi Tian Lui*, Kyle T Reid, Paula Tao* 4P Mahmoud Alnasleh*, Shuja Raza*, Sami Rahman*, Boyuan Zheng* 5P Amy Pochanart*, Matt Richardson, Peter Truong*, Jessica Wang 6P Maria-Elizabeth Baeva*, Andrew P. Golin*, Shivani Mysuria*, Priya Suresh* 7P Naomi Fettig*, Amanda Ryken*, Nicole Mar*, and Angela Sunario 8P Yasaman Jalalkamali, Niknaz Malekafzali*, Raisa Shabbir*, Tianna Sihota 9P Isabel G Jankowski 10P Wallace Yuen 11P Tristan T Lee 12P Leo T Liu 13P open 14P Brayden M. Wilkinson 15P Judy Ban 16P Leilynaz Malekafzali 17P Haya Abuzuluf 18P Yi Tian Liu*, May Dang-Lawson, Michael R. Gold

Presenting authors are denoted with an asterisk (*) following their name.

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Keynote Lecture

“B cell activation in space and time” Activated B cells secrete antibodies that protect against infection but which can also contribute to autoimmune diseases. Hence, the ability to modulate B cell activation, based on an understanding of the underlying mechanisms, has significant implications for health and disease. B cell activation is initiated by the binding of antigens to the B cell antigen receptor (BCR). The current focus of our lab is understanding how the cytoskeleton controls BCR mobility and spatial organization within the plasma membrane, and how that modulates BCR signaling and B cell activation. In particular, when B cells encounter antigens displayed on the surface of another cell, they form an immune synapse, a specialized membrane domain that amplifies BCR signaling. Our recent work has focused on identifying key regulators of immune synapse formation and function.

Dr. Mike Gold, Professor and Head

Department of Microbiology and Immunology University of British Columbia

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Group I - Bacteriology “Start Me Up” – The Rolling Stones

Oral Talks

Plasmid-Mediated Complementation of the Outer Membrane Capsule Biosynthesis Channel Gene wza May Restore Erythromycin Sensitivity in Escherichia coli K30

Authors: Karl Angelo Abuan*, Haya Abuzuluf*, Yuxin Ban*, Leilynaz Malekafzali* Course: MICB 421 Keywords: bacterial physiology, antibiotics Session: B-WESB

Isolated Supernatant Fraction of Escherichia coli K1 Inhibits Biofilm Formation of Wild Type Escherichia coli K12 Without Reducing Cell Viability.

Authors: Ian Lee, Felix Ma, Daniel Chan, Abigail Morres Course: MICB 421 Keywords: bacterial physiology, biofilm Session: A-MSL

Antibiotic-Induced Phage Resistance is not Unique to Gentamycin and Kanamycin but is a More General Response to Antibiotic Stress.

Authors: Wumian Wang, Myra Lam Course: MICB 447 Keywords: bacterial physiology, antibiotics Session: A-WESB

Sub-Lethal Exposure of Ampicillin and Gentamicin Does Not Confer Cross-Protection Against T7 Bacteriophage-Induced Lysis of Escherichia coli.

Authors: Amanda E Krystal*, Reiko Okamoto*, Andrea Sze*, Zachary F Weiss* Course: MICB 421 Keywords: bacterial physiology, bacteriophage biology Session: A-WESB

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RpoS-Mediated Stress Response Does Not Impact the Rate of T7 Bacteriophage-Induced Lysis of Escherichia coli

Authors: Kate W. Huang*, Erika L. Jang, Emily E. Kamma*, Annie H. Wu Course: MICB 421 Keywords: bacterial physiology, antibiotic resistance, stress response Session: A-WESB

Genetic Characterization and Investigation of Kanamycin Susceptibility of ompC and ompF Single Gene Deletion Mutants of Escherichia coli K-12.

Authors: Laurie Chang*, Jessie Zhang*, Jenna Ching, Regina Onal Course: MICB 421 Keywords: bacterial physiology, outer membrane proteins, genetics Session: B-MSL

Pseudo Finder: Automated Detection of Pseudogenes in Bacterial and Archaeal Genomes

Author: Mitch Syberg-Olsen Course: Keeling Lab Keywords: applied microbiology Session: B-WESB

Creation and Analysis of Caulobacter crescentus Whole Cell Bio-Catalysts for the Degradation of Lignocellulosic Biomass

Author: Tamar V. Av-Shalom* Course: directed studies, Hallam Lab Keywords: applied microbiology Session: B-WESB

Posters

Investigating the Relationship between Nitrofurantoin Sensitivity and Escherichia coli Group I Capsular Production by Studying its Biosynthesis Genes wza and wzb

Authors: Michelle Gu, Almas Khan, Desiree S. Pagulayan, Karen Tam Course: MICB 421 Keywords: bacterial physiology, antibiotic resistance Poster: 1P

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Investigating the Effects of Dibuteylethyl Phthalate (DEHP) on the Growth of Escherichia coli strain BL21

Authors: Jordan Brundrett, Jumar Orach, Lukas Sonderegger, Anthony Yan Course: MICB 421 Keywords: bacterial physiology Poster: 2P

OmpF Complementation Into an ompC/ompF Deficient Escherichia coli Strain Confers Sensitivity Towards SDS-EDTA Treatment

Authors: Vivian CW Fung*, Yi Tian Lui*, Kyle T Reid, Paula Tao* Course: MICB 421 Keywords: bacterial physiology, membrane proteins Poster: 3P

Sublethal Concentrations of Antibiotics Do Not Markedly Delay T7 Bacteriophage-Mediated Lysis Between Wild-Type, rpoS, and oxyR Mutants of Escherichia coli

Authors: Mahmoud Alnasleh*, Shuja Raza*, Sami Rahman*, Boyuan Zheng* Course: MICB 421 Keywords: bacterial physiology, bacteriophage Poster: 4P

Plasmid Mediated Complementation of wza in Escherichia coli K30 Strain CWG281 Restores Erythromycin Sensitivity

Authors: Amy Pochanart*, Matt Richardson, Peter Truong*, Jessica Wang Course: MICB 447 Keywords: bacterial physiology, genetics Poster: 5P

Plasmid-Mediated Overexpression of AcrS May Decrease Kanamycin Resistance in Escherichia coli

Authors: Maria-Elizabeth Baeva*, Andrew P. Golin*, Shivani Mysuria*, Priya Suresh* Course: MICB 447 Keywords: bacterial physiology, genetics Poster: 6P

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Growth Phase of Host Escherichia coli MG1655 Influences T4 and T7 Bacteriophage Replication Patterns During Coinfection

Authors: Naomi Fettig*, Amanda Ryken*, Nicole Mar*, and Angela Sunario Course: MICB 447 Keywords: bacterial physiology, bacteriophage biology Poster: 7P

The RcsB-dependent Upregulation of rprA Contributes to the Intrinsic Antibiotic Resistance of Escherichia coli Exposed to Antibiotics Targeting Cell Wall Synthesis but not Protein Synthesis

Authors: Yasaman Jalalkamali, *Niknaz Malekafzali, *Raisa Shabbir, Tianna Sihota Course: MICB 447 Keywords: antibiotic resistance Poster: 8P

Characterization and Gene Transcription of Flavin Transferases in Desulfitobacterium hafniense

Author: Isabel G Jankowski Course: co-op Keywords: gene expression Poster: 9P

Characterizing Localization and Protein-Protein Interactions of the Enteropathogenic Escherichia coli Kinases NleH1 and NleH2

Author: Tristan T Lee* Course: directed studies, Finlay Lab Keywords: bacterial pathogenesis Poster 11P:

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Group II - Immunology “With a Little Help from My Friends” – The Beatles

Oral Talks

Intestinal Epithelial Inflammasomes: Frontline Defenders Against Campylobacter jejuni Infection.

Author: Thomas Hoang Course: directed studies, Vallance Lab Keywords: host pathogen interactions, bacterial pathogenesis Session: A-MSL

Liposomal Formulations of Host Defense Peptides Retain Efficacy While Reducing Cytotoxicity and Aggregation

Author: Leo T Liu Course: directed studies, Hancock Lab Keywords: innate immunity, antibiotics Session: A-MSL

Survival of Caenorhabditis elegans Infected with Escherichia coli DFB1655 is not Affected by a Missense Mutation in dop-1 or Treatment with Chlorpromazine Hydrochloride

Author: Chris James, Ofodile Morah, Victoria Panwala, Amin Yarmand* Course: MICB 421 Keywords: innate immunity, host pathogen interactions Session: B-MSL

The Role of Tumor Necrosis Factor Alpha (TNF) Receptor CD120b Expression on Human Regulatory T cell (Treg) Proliferation Using CRISPR-Cas Mediated Knockout

Author: Uyen Nguyen Course: directed studies, Levings Lab Keywords: cytokines, adaptive immunity Session: A-WESB

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Low Affinity FcγRIIIA Promotes IVIg-Mediated IL-10 Production in Monocyte Derived Macrophages

Author: Jordan Brundrett Course: directed studies, Sly Lab Keywords: cytokines, adaptive immunity Session: A-MSL

ADAM8 Potentiates Trophoblast Migration by Controlling 1 Integrin-Mediated Cell Adhesion

Author: HT Le, J Atif*, DL Mara, B Castellana, J Treissman, AG Beristain. Course: directed studies, Beristain Lab Keywords: signalling Session: B-MSL

Aryl Hydrocarbon Receptor Nuclear Translocator-2 Expression Characterizes the Remyelinating and Repair Potential of Oligodendrocytes in a Model of Multiple Sclerosis

Author: Jake Johnston*, Pierre Becquart, Jacqueline Quandt Course: directed studies, Quandt Lab Keywords: disease modeling Session: A-MSL

Posters

Bacterial Exotoxin Sequestration with Engineered Liposomes

Author: Leo T Liu Course: co-op, Hancock Lab Keywords: innate immunity, lipids, biotechnology Poster: 12P

The Role of Macrophages and Neutrophils During -cell Development and Regeneration Post-injury in Zebrafish

Authors: Brayden M. Wilkinson Course: directed studies, Kieffer Lab Keywords: model systems, immunity Poster: 14P

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Inhibition of LIM Kinase (LIMK) Impairs B Cell Receptor-Induced B Cell Spreading by Causing Hyperactivation of the Actin-Severing Protein Cofilin

Authors: Yi Tian Liu*, May Dang-Lawson, Michael R. Gold Course: directed studies, Gold Lab Keywords: B cells, cell biology Session: 18P

The Role of STAT1 in Coordinating CD8+ T cell-mediated Responses to Murine Norovirus

Author: Wallace Yuen Course: directed studies, Osborne Lab Keywords: gene expression Poster: 10P

Group III - New Concepts in Virology

“We Will Rock You” - Queen

Oral Talks

Biological Individuality: The Diversity of Intestinal Virome Onsets Type I Diabetes in Genetically Susceptible Children

Author: Niknaz Malekafzali Course: MICB 406 Keywords: Microbiome-Virome Interactions Session: B-WESB

A Cure for Cancer? The Therapeutic Potential of Combining Oncolytic Viruses and Chimeric Antigenic Receptor T-Cell Therapy

Author: Andrew P. Golin Course: MICB 406 Keywords: Viruses and Cancer Session: B-WESB

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Salivary Exosomal microRNAs: Discovery and Potential Biomarkers for Human Papillomavirus- Associated Oral Squamous Cell Carcinoma

Author: Syed S Raza Course: MICB 406 Keywords: Viruses and Cancer Session: B-WESB

A Double-Edged Sword: The Therapeutic Potential of Chimeric Antigen Receptor T Cell in HBV/HIV Co-Infected Patients

Author: Boyuan Zheng Course: MICB 406 Keywords: Prevention of and Therapy for Viral Infections Session: B-MSL

Discovery and Characterization of Blood-Brain Barrier Targets to Develop New Therapeutic Strategies to Treat Neurotropic Viral Infections

Author: Felix Ma Course: MICB 406 Keywords: Prevention of and Therapy for Viral Infections Session: A-WESB

Posters

Targeted Delivery of Therapeutic Exosome to HIV-1 Reservoir Cells: A New Strategy to HIV-1 Eradication in Children

Author: Judy Ban Course: MICB 406 Keywords: Prevention of and Therapy for Viral Infections Poster: 15P

Emerging Role of Circulating MicroRNAs and Opioid-mediated Enhancement of HIV-1 Infection in People Who Inject Drugs (PWID): Opportunities for Biomarkers and Novel Antiviral Strategies

Authors: Leilynaz Malekafzali Course: MICB 406 Keywords: Prevention of and Therapy for Viral Infections Poster: 16P

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Group IV - Biology of Infection Across Diverse Viral Systems

“Stayin’ Alive” – The Bee Gees

Oral Talks

MERS-CoV: Identifying Viral and Host Factors Associated with Transmission, Pathogenesis, and Treatment Development

Author: Sami Rahman Course: MICB 406 Keywords: Virus-host interactions Session: B-MSL

Crossing barriers: How John Cunningham Virus Crosses the Blood Brain Barrier and Induces Progressive Multifocal Leukoencephalopathy in Patients on Disease-Modifying Therapies.

Author: Maria-Elizabeth Baeva Course: MICB 406 Keywords: Virus-host interactions Session: A-WESB

Leishmania RNA Virus: Mere Endosymbiont or Major Player in Leishmania-associated Disease?

Authors: Shivani Mysuria Course: MICB 406 Keywords: Virus-host interactions Session: B-MSL

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Zika Virus Infection: a Cause or Explanation in the Development of Guillain-Barre Syndrome?

Authors: Priya Suresh Course: MICB 406 Keywords: Virus-host interactions Session: A-MSL

Posters

Human Endogenous Retroviruses as Contributors of Exosome-mediated Autoimmunity in Systemic Lupus Erythematosus

Authors: Haya Abuzuluf Course: MICB 406 Keywords: Virus-host interactions Poster: 17P

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Abstracts Group I: Bacteriology Session B-WESB (4:00 – 5:15pm) Authors: Karl Angelo Abuan*, Haya Abuzuluf*, Yuxin Ban*, Leilynaz Malekafzali* Plasmid-Mediated Complementation of the Outer Membrane Capsule Biosynthesis Channel Gene wza May Restore Erythromycin Sensitivity in Escherichia coli K30 The assembly and transport of type 1 capsular polysaccharide in Escherichia coli K30 is mediated through the Wzy-dependent pathway. This pathway involves: Wza, an outer membrane channel; Wzb, a cytosolic phosphatase; and Wzc, an autokinase in the inner membrane. Previous research has shown that deletion of Wza confers resistance to erythromycin. It has been suggested that the route of entry of erythromycin is partially dependent on the Wza channel. In this study, we hypothesize that plasmid-mediated complementation of wza will restore erythromycin sensitivity in a wza E. coli knockdown mutant. The wza was previously cloned into an arabinose-inducible pBAD24 vector and transformed into the wza knockdown mutant. Using a modified Kirby-Bauer disk diffusion assay, 1% arabinose was observed to induce optimal pBAD24-wza expression without impairing cell growth. We found that the wza knockdown containing pBAD24-wza showed significantly higher erythromycin sensitivity in comparison to the control strain without the plasmid. Our findings indicate that plasmid-mediated complementation of wza restores erythromycin sensitivity in the wza knockdown mutant strain. Course: MICB 421

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Group I: Bacteriology Session A-MSL (1:30 – 2:45pm) Authors: Ian Lee, Felix Ma, Daniel Chan, Abigail Morres Isolated Supernatant Fraction of E. coli K1 Inhibits Biofilm Formation of Wild Type E. coli K12 Without Reducing Cell Viability. Previous studies have indicated that group 2 capsular polysaccharides isolated from extracellular fractions of uropathogenic E. coli strains are able to exhibit broad spectrum inhibition of biofilm formation in commensal strains of E. coli without impacting cell viability. We hypothesized that isolated supernatant from a non-pathogenic lab strain of E. coli known to produce group 2 polysaccharides would be sufficient to reduce biofilm formation in a wild type E. coli strain. To test this, we used an optimized biofilm formation assay to quantify and compare biofilm formation of the wild type strain treated with and without capsule-containing supernatant in minimal glucose media. We found that both crude and filtered supernatants of the non-pathogen E. coli were able to significantly reduce the degree of biofilm formation without adversely impacting cell viability. As such, we are able to provide indication for the use of processed supernatants from non-pathogen E. coli as a potential tool towards the study of biofilm regulation through its inhibitory effect in the context of minimal biosafety risk levels. Course: MICB 421

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Group I: Bacteriology Session A-WESB (1:30 – 2:45pm) Authors: Wumian Wang, Myra Lam Antibiotic-Induced Phage Resistance is Not Unique to Gentamycin and Kanamycin, but is a More General Response Among Other Antibiotics. Bacteriophage has long been researched and investigated not only as a research tool, but also as a medical treatment to treat bacterial infections. Although phage therapy has not been used as extensively as antibiotic treatments, phage therapies have massive potential in clinical settings, and are currently being researched as future treatment possibilities on a large scale. Thus, the interaction between phage and bacteria show a fascinating and important relationship in potential medical applications, and any synergistic or contraindicating effects could be of importance to the use of such treatments, perhaps in conjunction with each other. In this study, we demonstrated that sub-lethal treatments of Escherichia coli B23 with the beta-lactam antibiotics ampicillin and penicillin, and aminoglycoside antibiotics gentamicin and kanamycin lead to decreased infectivity of T7 phage, using a real-time cell lysis assay. Incubation of E. coli in the exponential growth phase with T7 phage showed that antibiotic-treated groups require longer incubation times to reach a decline in OD compared to the non-treated group. This result suggests that the antibiotic-induced phage resistance is not unique to one class of antibiotics, and might belong to a general response to stress. Course: MICB 447

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Group I: Bacteriology Session A-WESB (1:30 – 2:45pm) Authors: Amanda E Krystal*, Reiko Okamoto*, Andrea Sze*, Zachary F Weiss* Sub-Lethal Exposure of Ampicillin and Gentamicin Does Not Confer Cross-Protection Against T7 Bacteriophage-Induced Lysis of E. coli. In order to respond to stress, bacteria like E. coli employ robust sensing systems to regulate gene transcription. One such regulator, the sigma factor RpoS, has been shown to be upregulated in response to stress, including antibiotics. Furthermore, studies have shown that exposure to a stressor may lead to increased tolerance to a subsequent stressor in a phenomenon known as cross-protection. A recent investigation demonstrated this phenomenon, whereby E. coli cells treated with sub-lethal concentrations of antibiotics exhibited delayed T7 bacteriophage-induced lysis compared to untreated cells. Here, we re-examine cross-protection and the role of RpoS in response to sub-lethal concentrations of antibiotics. Using an infectivity assay in a 96-well plate, cells pre-treated with a gradient of sub-lethal concentrations of ampicillin or gentamicin were incubated with T7 and optical density was recorded over time to track lysis rates. Contrary to previous studies, delayed lysis was not observed in antibiotic-treated wild-type cells, suggesting the absence of cross-protection. Strikingly, however, cells treated with lower-end concentrations of ampicillin displayed earlier T7-induced lysis than untreated cells, indicating the potential for an ampicillin-specific pathway affecting the T7 lytic cycle. Furthermore, performing this infectivity assay with rpoS knock-out mutant cells resulted in no notable difference compared to wild-type, demonstrating that RpoS does not play a significant role in mediating the response to sub-lethal concentrations of antibiotics. Additionally, a Western blot using anti-RpoS antibody was conducted to investigate the relative levels of RpoS expression in wild-type and rpoS mutant cells following treatment with similar antibiotic concentrations. While this assay was able to confirm the presence and absence of RpoS in wild-type and mutant cells, respectively, results for wild-type cells treated with sub-lethal antibiotics were inconclusive. Together, these data illustrate that the previously observed phenomenon of cross-protection may be inaccurate in this context, and also indicate that the presence of RpoS does not play a significant role. Course: MICB 421

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Group I: Bacteriology Session A-WESB (1:30 – 2:45pm) Authors: Kate W. Huang*, Erika L. Jang, Emily E. Kamma*, Annie H. Wu RpoS-Mediated Stress Response Does Not Impact the Rate of T7 Bacteriophage-Induced Lysis of Escherichia coli Exposure to environmental stressors causes activation of the alternative sigma factor, RpoS, in Escherichia coli. This regulatory protein modulates the expression of general stress response genes and can induce cross-protection against secondary environmental stressors. Previous studies suggested that treatment with sub-lethal concentrations of antibiotics as a primary stressor can delay T7 bacteriophage-induced lysis. We hypothesized that the delayed lysis observed in Escherichia coli pre-treated with antibiotics is the result of cross-protection conferred by RpoS activation, with T7 bacteriophage infection as the secondary stressor. We removed the kanamycin resistance cassette from an rpoS knockout (rpoS) mutant using FLP-FRT recombination, and we used minimum inhibitory concentration assays to determine the sub-lethal kanamycin and ampicillin concentrations for the rpoS mutant and wild-type cells. After pre-treating the cells with sub-lethal concentrations of antibiotics, we performed an OD-based real-time T7 bacteriophage infectivity assay to generate lysis curves of each treatment (n=3). The rpoS mutant exhibited similar lysis curves to the wild-type cells. Contrary to previous findings, our results suggest that pre-treatment with sub-lethal concentrations of kanamycin and ampicillin does not delay T7 bacteriophage-induced lysis in wild-type cells. This indicates that the RpoS-mediated general stress response may not confer cross-protection against T7 bacteriophage infection. Course: MICB 421

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Group I: Bacteriology Session B-MSL (4:00 – 5:15pm) Authors: Laurie Chang*, Jessie Zhang*, Jenna Ching, Regina Onal Genetic Characterization and Investigation of Kanamycin Susceptibility of Ompc and Ompf Single Gene Deletion Mutants of Escherichia coli K-12. OmpF and OmpC are trimeric outer membrane proteins of gram-negative bacteria that form pores containing 16-stranded beta-barrels. The two porins are general diffusion porins that have a central hydrophilic core. Negatively charged residues are found on the L3 loop of the Omp proteins, while positively charged residues are clustered on the opposite barrel wall. The OmpF and OmpC porins have been shown to transport a variety of antibiotics. Thus, the loss of OmpF and OmpC expression has been implicated in the development of antibiotic resistance. Much remains unknown about aminoglycoside transport through the OmpF and OmpC porins of gram-negative bacteria. Previous studies relating the OmpF porin to kanamycin resistance have been inconclusive. OmpC is more cation selective than OmpF, which suggests OmpC may be more involved in the entry of polycationic kanamycin into the cell than OmpF, however OmpF has a larger porin diameter which may result in increased drug translocation into the cell. To investigate the role of the porins in kanamycin susceptibility, Escherichia coli K-12 ompC and ompF single gene deletion mutants were obtained from the Keio collection. Previous papers have found that the Keio ompF deletion mutant still had and expressed the ompF gene. To avoid the same error, we performed genetic characterization of the ompC and ompF single deletion mutants from the Keio collection before and after removal of kanamycin resistance cassette from the site of the deleted gene. FLP-FRT recombination was performed to remove the kanamycin resistance cassette from the Keio strains, while minimum inhibitory concentration assays were used to determine kanamycin susceptibility of mutants. We have shown the gene deletion mutations are accurate and precise in our Keio mutants, and we have preliminary results showing that OmpF deletion mutant has a greater resistance to kanamycin, as compared to wildtype and the OmpC deletion mutant. This finding implicates that ompF may play a greater role in kanamycin susceptibility. Course: MICB 421

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Group I: Bacteriology Session B-WESB (4:00 – 5:15pm) Authors: Mitch Syberg-Olsen Pseudo Finder: Automated Detection of Pseudogenes in Bacterial and Archaeal Genomes. Pseudogenes are DNA sequences that were once functional genes but have lost functionality due to mutations. While it is commonly thought that prokaryotic genomes are highly space efficient and therefore would not contain many pseudogenes, there are many cases where this is not true. Microbes that have experienced lifestyle shifts over evolutionary time, particularly those that have become dependent on other organisms (pathogens or symbionts), can have up to half of their genes pseudogenized. These pseudogenes must be considered, otherwise the true functional capabilities of the microbe cannot be understood. In addition, pseudogenes contain information about the evolutionary history of the organism. Despite the importance of pseudogenes, there are no current open-source tools for prokaryotic pseudogene annotation. Pseudo finder is a command-line tool for pseudogene detection in prokaryotic genomes. It is open source and has fully customizable parameters, so it can be fine-tuned for any situation. Pseudo finder locates truncated and fragmented genes using BLAST and can even recover highly degraded pseudogenes from areas of the genome that lack predicted open reading frames. A standard 2 megabase genome can be annotated in an hour, and if you decide to change settings, subsequent passes on the same genome take only 30 seconds. The rapid annotation speed of this program opens the door for pseudogene analyses that were previously not possible and also provides a method for standardizing prokaryotic pseudogene annotation. Pseudo finder has been used to annotate pseudogenes in 15 genomes of obligate symbionts. The genomes ranged from 4-20% pseudogenized, which could be reflective of their different evolutionary histories. Customizable parameters allowed for annotation of organisms ranging from obscure symbionts to E. coli, which has been shown in literature to have approximately 185 pseudogenes. Pseudo finder was also used in one case to provide evidence that an organism identified in a next-generation sequencing library was an unexpected symbiont. This tool provides a rapid, reproducible and easy solution for an important problem in prokaryotic genome analysis. Course: Keeling Lab

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Group I: Bacteriology Session B-WESB (4:00 – 5:15pm) Author: Tamar V. Av-Shalom Creation and Analysis of Caulobacter crescentus Whole Cell Bio-Catalysts for the Degradation of Lignocellulosic Biomass. Todays modern society depends on the use of fossil fuels for the creation of a wide variety of chemicals and polymers used daily. While advances in sequencing technology and recombinant DNA manipulation has made it possible for us to use microbes to synthesize equivalent products through biosynthetic pathways, it remains difficult for us to use these engineered microbes in a carbon neutral way. Lignocellulosic biomass represents a promising feedstock for bioprocessing. The recalcitrant nature of the lignocellulose polymer represents a technical barrier for the full exploitation of this resource. One alternative to traditional and expensive methods to breakdown lignocellulose this is the process of consolidated bioprocessing, where microbes will facilitate both the breakdown of the lignocellulose and its conversion into useful compounds. Engineering new microbes capable of degrading lignocellulosic biomass is essential for creating efficient new strains for consolidated bioprocessing. In this study we examine the design, engineering, and characterization of Caulobacter crescentus whole-cell biocatalysts capable of degrading lignocellulosic biomass. We attempt to use methods of both surface display and secretion in C. crescentus to engineer strains that can degrade lignin. Additionally, we characterize previously cloned strains of C. crescentus displaying five different cellulase enzymes representing a wide range of superfamilies of glycoside hydrolases with different specificities when hydrolysing the release of glucose from cellulose. We characterize the activity of these enzymes as well as the ability of C. crescentus displaying these enzymes to support the growth of another bacterium in consortium. This study has shown that by using recombinant DNA technologies we can insert functionally active enzymes into the S-layer of C. crescentus creating fusion proteins that are displayed on the surface of the cell. Additionally, we show that these active enzymes add a fitness benefit to C. crescentus, allowing it to grow on defined media supplemented only with carboxyl methylcellulose. Course: directed studies Hallam Lab

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Group I: Bacteriology Poster MSL 101-1P Authors: Michelle Gu, Almas Khan, Desiree S. Pagulayan, Karen Tam Investigating the Relationship between Nitrofurantoin Sensitivity and Escherichia coli Group I Capsular Production by Studying its Biosynthesis Genes wza and wzb. The group I capsular polysaccharides (CPS) is assembled through the Wzy-dependent pathway that involves Wza, Wzb and Wzc proteins. It has been characterized as a virulence factor that confers antimicrobial resistance in Escherichia coli (E. coli). Increasing antimicrobial resistance among uropathogens has led to increased interest in using the antibiotic nitrofurantoin. While previous studies have investigated the effect of single deletion of wza, wzb, and wzc genes on erythromycin sensitivity in E. coli K30 E69, susceptibility of these individual gene knockout strains to nitrofurantoin has not yet been explored. We aimed to investigate the relationship between nitrofurantoin sensitivity and E. coli group I CPS production by studying its biosynthesis genes wza and wzb. Strains were identified via antibiotic sensitivity testing and PCR. Zones of inhibition for each strain were quantified via a disc diffusion assay, and nitrofurantoin minimum inhibitory concentrations (MIC) were determined. Results of the disc diffusion assay revealed significant differences in zone of inhibition sizes for the full knockout and wzb mutant strain relative to wild type (WT). In contrast to K30 wzb mutant, the K-12 wzb mutant from Keio collection showed less sensitivity than the WT parent strain. We conclude that other factors besides capsule may be contributing to nitrofurantoin susceptibility, and that Wzb may be involved in complex interactions with substrates that affect downstream nitrofurantoin activation. Course: MICB 421

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Group I: Bacteriology Poster MSL 101-2P Authors: Brundrett, Jordan; Orach, Juma; Sonderegger, Lukas; Yan, Anthony Investigating the Effects of Dibuteylethyl Phthalate (DEHP) on the Growth of Escherichia coli strain BL21. Phthalates (also known as phthalate esters) are organic chemicals used as plasticizers in a wide range of food packaging, containers, and other household plastic objects. Humans are exposed to phthalates through the air and by ingestion. Phthalate exposure has been shown to induce changes to the microbiome, causing shifts in the resident microbiota in a dose-dependent manner. One phthalate in particular, dibuteylethyl phthalate (DEHP), is known to be present at high concentrations in Canadian food packaging. Previous research has found that DEHP has biphasic effects on the growth of E. coli. To further investigate the effect DEHP may have on E. coli growth, our research set out to determine the effects a dose-dependent exposure on this bacterium. To do this, minimum inhibitory concentration (MIC) assays were performed, disc diffusion assays, as well as growth curves. Initial MIC assays showed that DEHP does not have a dose-dependent growth-inhibitory effect on E. coli. However, phase separation between the DEHP solution and E. coli culture media made it difficult to determine whether growth inhibition was due to the DEHP or to inadequate aeration of the growing culture. Disc diffusion assays indicated DEHP did not have a growth-inhibitory effect on E. coli. The addition of ethanol reduced the phase separation between DEHP and E. coli culture media. However, subsequent growth curves showed conflicting results which suggested that the addition of ethanol may be the reason for reduced growth rate. Course: MICB 421

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Group I: Bacteriology Poster MSL 101-3P Authors: Vivian CW Fung*, Yi Tian Lui*, Kyle T Reid, Paula Tao* OmpF Complementation into an ompC/ompF Mutant of Escherichia coli Confers Membrane Sensitivity Towards SDS-EDTA Treatment. The outer membrane (OM) on gram negative bacteria contributes to inherent antibiotic resistance as well as protection against toxins. OM general diffusion porins, such as OmpC and OmpF, regulate movement of biomolecules in and out of the periplasm, preventing large and potentially harmful molecules from entering the bacteria. OmpC and OmpF have also been shown to contribute to membrane stability by maintaining membrane asymmetry through the Mla pathway. Previous studies have found deletion of OmpC (ompC) have sensitized Escherichia coli towards the detergent sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). In contrast, double deletions of ompC and ompF show comparable resistance to wild-type (WT) E. coli. Further studies into single ompF deletions have show no appreciable difference in sensitivity to SDS-EDTA from wild-type. It is our hypothesis that expression of OmpF in the absence of OmpC will induce the sensitive phenotype towards SDS-EDTA. To test this, we transformed a vector containing ompF into ompC/ompF mutants, then compared their relative sensitivity to WT and ompC mutants over increasing EDTA concentrations. Our study found that complementation of ompF back into ompC/ompF mutants induced an increased sensitivity towards SDS-EDTA. Our results implicate a dynamic relationship between OmpF and OmpC, where presence of OmpC and expression of OmpF may influence OM stability. Course: MICB 421

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Group I: Bacteriology Poster MSL 101-4P Authors: Mahmoud Alnasleh*, Shuja Raza*, Sami Rahman*, Boyuan Zheng* Sublethal Concentrations of Antibiotics Do Not Markedly Delay T7 Bacteriophage-Mediated Lysis Between Wild-Type, rpoS, and oxyR Mutants of Escherichia coli T7 bacteriophages are obligatory lytic phages that attach to the lipopolysaccharide (LPS) on the outer membrane (OM) of host cells, and inject their DNA into host cell cytoplasm. By hijacking the cellular machinery in the host cell, the bacteriophage can replicate their genome and proliferate. Bacteriophage infection can act as an environmental stressor for E. coli, similar to other known factors such as antibiotics, heat, and nutrient deprivation. In response to stressors, E. coli has been shown to increase the production of certain regulatory proteins such as RpoS and OxyR. RpoS is an RNA polymerase sigma factor responsible for regulating genes necessary for the general stress response, while OxyR is the principal regulator for hydrogen peroxide detoxification in E. coli cells. Both regulators may play a role in protective response against oxidative stress, which is induced by exposure to sublethal concentration of antibiotics. Previous studies have suggested that increased production of RpoS and OxyR following sublethal concentration of antibiotics may play an important role in delaying T7 bacteriophage-mediated cell lysis. However, such cross-protection phenomenon is not well-understood. The aim of our project is to investigate the role of RpoS and OxyR in delaying T7 bacteriophage-mediated cell lysis following exposure to sublethal concentrations of antibiotics by comparing the onset of lysis in E. coli WT (wild-type) strains to that of rpoS and oxyR single knockout mutant strains. We hypothesized that both RpoS and OxyR are necessary for the delayed T7 bacteriophage-mediated cell lysis in E. coli strain BW25113 following exposure to sublethal concentrations of antibiotics. Validation of the WT and the two mutants was achieved via PCR and sublethal concentration of ampicillin, gentamicin, and kanamycin were determined using a Minimum Inhibitory Concentration (MIC) assay. Phage infectivity assay of the two mutant E. coli strains treated with a range of sublethal ampicillin did not show a change in the onset or the rate of lysis compared to the WT. Similar infectivity assay result was observed in the gentamicin treatment. Course: MICB 421

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Group I: Bacteriology Poster MSL 101-5P Authors: Amy Pochanart*, Matt Richardson, Peter Truong*, Jessica Wang Plasmid Mediated Complementation of Wza in Escherichia coli K30 Strain CWG281 Restores Erythromycin Sensitivity. In Escherichia coli serotype K30, the outer membrane channel protein Wza, involved in capsular polysaccharide secretion, has been genetically linked to macrolide sensitivity. Deletion of wza in erythromycin-sensitive E. coli K30 confers resistance to the antibiotic. In this study, we investigated whether complementation of wza restores sensitivity to erythromycin in the wza knockout mutant E. coli CWG281. Using Gibson assembly, wza was cloned into the arabinose-inducible pBAD24 expression vector and then transformed into E. coli CWG281. Induction of pBAD24-wzaK30 with 0.008% arabinose was observed to restore erythromycin sensitivity, assessed using Kirby-Bauer disk diffusion assays. Interestingly, induction of pBAD24-wzaK30 with 1% arabinose was found to impair growth of E. coli CWG281 transformants. From this study, we conclude that complementation of wza in E. coli CWG281 is sufficient to restore erythromycin sensitivity. We further conclude that overexpression of wza in E. coli CWG281 impairs cell growth. Course: MICB 447

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Group I: Bacteriology Poster MSL 101-6P Authors: Maria-Elizabeth Baeva*, Andrew P. Golin*, Shivani Mysuria*, Priya Suresh* Plasmid-Mediated Overexpression of AcrS may Decrease Kanamycin Resistance in Escherichia coli. Escherichia coli encodes efflux pumps that confer resistance to antimicrobial compounds. Previous studies have shown that efflux pumps AcrAB and AcrEF have similar structures and functions, and may be repressed by homologous transcriptional repressors. The transcriptional repressor AcrR, represses the acrAB operon. A gene encoding a less-studied putative transcriptional repressor AcrS, and acrR are both located upstream of acrEF and acrAB, respectively. Deleting acrS in Escherichia coli has also shown to result in increased levels of acrE mRNA when cultured in the presence of sub-inhibitory concentrations of kanamycin. Given this evidence that AcrS may repress acrE, we hypothesize that increasing AcrS expression will result in increased repression of acrE and confer a decrease in kanamycin resistance. An inducible expression vector containing acrS was constructed and transformed into wild-type E. coli strain BW25113, as well as strains bearing deletions of acrS or acrE. Resistance to kanamycin was tested using a minimum inhibitory concentration assay. Results from this study suggest that overexpressing AcrS decreased kanamycin resistance in E. coli BW25113. This supports the hypothesis that AcrS may be a transcriptional repressor of acrE in E. coli BW25113. Course: MICB 447

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Group I: Bacteriology Poster MSL 101-7P Authors: Naomi Fettig*, Amanda Ryken*, Nicole Mar*, and Angela Sunario Growth Phase of Host Escherichia coli MG1655 Influences T4 and T7 Bacteriophage Replication Patterns During Coinfection. T4 and T7 bacteriophages have been proposed to compete during coinfection of Escherichia coli, impacting their replication patterns relative to monoinfection conditions. In monoinfection conditions, T4 has been suggested to replicate optimally in exponential phase cells, while T7 in stationary phase cells. Our study investigates whether similar replication patterns will be observed in coinfection conditions by inoculating T4 and T7 phages at multiplicity of infection of 3 in two growth phases of E. coli MG1655: early exponential and stationary. The replication patterns of both phages were analyzed by quantitative real-time Polymerase Chain Reaction performed on samples from each culture condition collected over a period of six hours following infection. We expected T4 to exhibit greater similarity of replication pattern in coinfection relative to monoinfection conditions than T7 in early exponential phase, and vice versa for stationary phase. Our data supports the hypothesis, where T4 achieves a higher relative copy number in the early exponential phase. Although both phages show reduced copy number in the stationary phase, T7 exhibits less of a reduction than T4, suggesting that it replicates more effectively at this later growth phase. Our results indicate that competitive interactions between T4 and T7 bacteriophages are indeed impacted by the growth phase of the host cells. Course: MICB 447

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Group I: Bacteriology Poster MSL 101-8P Authors: Yasaman Jalalkamali, Niknaz Malekafzali, Raisa Shabbir, Tianna Sihota. The RcsB-dependent Upregulation of rprA Contributes to the Intrinsic Antibiotic Resistance of Escherichia coli Exposed to Antibiotics Targeting Cell Wall Synthesis but not Protein Synthesis. The regulation of capsular synthesis (Rcs) phosphorelay system allows Escherichia coli to respond to stress resulting from disruption of the peptidoglycan layer. The cytosolic response regulator, RcsB, regulates the expression of various downstream genes including rprA. rprA is a small non-coding RNA that stabilizes the translation of RpoS, a regulator of general stress response in E. coli. Previous studies have shown that an E. coli K-12 strain bearing a deletion of rcsB displays enhanced susceptibility to -lactams, which target peptidoglycan synthesis. This result correlated with a decrease in rprA expression. It is not known whether induction of rprA expression is limited to -lactams. The objective of this study was to determine rprA expression levels using -galactosidase assays in both wild-type and delta-rcsB, following treatment with sub-inhibitory concentrations of antibiotics targeting either peptidoglycan or protein synthesis. We hypothesized that following treatment with -lactams, rprA expression would be lower in a strain bearing a deletion of rcsB in comparison to the wild-type strain. Furthermore, within a given strain, we expected rprA expression to be upregulated in response to -lactam stress and unchanged in response to aminoglycoside stress in comparison to unstressed (no antibiotic) conditions. Antibiotics that target cell wall synthesis and would induce the Rcs phosphorelay system while antibiotics that target protein synthesis and would not activate Rcs via peptidoglycan disruption. We observed that in the absence of rcsB, the minimum inhibitory concentration decreased two-fold with respect to treatment with penicillin and phosphomycin but remained unchanged when treated with tetracycline and streptomycin. rprA expression was upregulated in wild-type cells in the presence of -lactams or aminoglycosides and was suppressed in the absence of rcsB when treated with either -lactams or aminoglycoside antibiotics. This study provides insight into the intrinsic Rcs-mediated stress response mechanisms used by E. coli through the expression of RcsB. Course: MICB 447

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Group I: Bacteriology Poster MSL 101-9P Author: Isabel G Jankowski Characterization and Gene Transcription of Flavin Transferases in Desulfitobacterium hafniense. Tetrachloroethene (PCE) is a common soil and groundwater pollutant that is recalcitrant under

aerobic conditions. Under anaerobic conditions, certain microorganisms, such as

Desulfitobacterium hafniense, are able to degrade PCE by using it as an electron acceptor. While

the main catalytic subunit in the process, PceA, has been well studied, the PceC protein remains

to be characterized, and represents the best candidate for electron transfer to PceA. Production

and characterization of PceC is a challenge due to its six transmembrane domains and flavin

mononucleotide (FMN) cofactor requirement. Preliminary in vitro data showed that it is possible

to reconstitute the exocytoplasmic FMN-binding domain of PceC (PceC-FBD) with the help of a

flavin-transferase protein (Ftp) found in D. hafniense. To further investigate the role of Ftp in the

context of PceC and reductive dechlorination, two goals were targeted: (i) to develop a simple,

replicable assay to characterize Ftp, and (ii) to measure transcriptional levels of ftp1, ftp2, and

pceC under various culture conditions in search of regulatory patterns. To complete the first

goal, a 96-well plate assay was developed by mixing pure Ftp1, pure urea-denatured PceC-FBD,

Mg2+, and flavin adenine dinucleotide (FAD). Reconstituted PceC-FBD with covalently bound

FMN was used as a proxy for Ftp1 activity, and was detected in a protein gel by UV

illumination. Results demonstrated that our assay was functional, however needs to be optimized

for stronger signal and reduced protein precipitation. To complete the second goal, D. hafniense

strain LBE was cultured using H2/PCE, and lactate/fumarate, as respective electron

donors/acceptors. A third culture was grown on lactate/fumarate, and spiked with PCE after 22 h

of growth, to see if the presence of PCE would change transcription levels. Cell densities were

tracked over time by optical density, and RNA was extracted for RT-qPCR analysis of ftp1, ftp2,

and pceC genes, at select time points. Results showed that ftp genes are not co-regulated with

pceC. It was also found that the addition of PCE to the culture grown on lactate/fumarate caused

noticeable stress on growth, and did not affect the transcriptional levels of the three genes. These

RNA results suggest that Ftp activity may not be specific for PceC. Ftp does, however, catalyze

the assembly and maturation of PceC-FBD. This relationship and methodology can be used to

characterize Ftp proteins in D. hafniense, as demonstrated, and to help further our understanding

of reductive dechlorination.

Course: International research internship at EPFL, Switzerland

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Group I: Bacteriology Poster MSL 101-11P Author: Tristan T Lee* Characterizing Localization and Protein-Protein Interactions of the Enteropathogenic Escherichia coli Kinases NleH1 and NleH2. Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal-causing pathogen that colonizes the human intestinal epithelium. During infection, the kinase effectors NleH1 and NleH2 are secreted through the type III secretion system into host cells. Despite sharing a high sequence homology of 84%, NleH1 supresses, whereas NleH2 stimulates, the NFκB-mediated inflammatory response. The specific mechanisms, interacting partners, and kinase targets of NleH1/2 are not well characterized. Kinase substrate specificity is often dictated by subcellular localization. Kinases may be targeted to membranes by addition of lipophilic groups like myristic or palmitic acid. NleH1 has recently been shown to localize to the plasma membrane, and sequence analysis has predicted the N-terminii of both NleH1/2 to have putative myristoylation and palmitoylation sites. In most cases, eukaryotic enzymes sequential add myristoyl and palmitoyl groups to glycine and cysteine, respectively, which I hypothesize are exposed upon cleavage of the secretion signal. To investigate this further, the first 20 amino acids of NleH1/2 wild-type or double alanine mutant were fused to β-lactamase and localization was assessed through infection by subcellular fractionation to separate cytosolic from membrane proteins and visualized by fluorescence microscopy. A dynamic mechanism of regulating biological activity is through protein-protein interactions, where modular domains bind to short recognition sequences on target proteins. In the case of NleH1/2, the C-terminii contain a PDZ binding motif (PBM), which interacts with PDZ on the scaffold protein, NHERF2. Prior research has not determined whether the translocation of this domain impacts interaction. By creating a series of mutants changing the position of the C-terminal PBM, I have demonstrated that these domains are essential for protein stability of NleH1/2. The interaction of NleH with NHERF2 upon EPEC infection of HeLa cells has been assessed by co-immunoprecipitation. These experiments further our understanding of the effect of localization and protein-protein interaction on the complex interplay between pathogens and host cell signaling responses. Course: directed studies, Finlay Lab

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Group II: Immunology Session A-MSL (1:30 – 2:45pm) Author: Thomas Hoang Intestinal Epithelial Inflammasomes: Frontline Defenders Against Campylobacter jejuni Infection. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Commonly acquired through consumption of contaminated water or poultry, it triggers symptoms of cramps, fever and bloody diarrhea. Past research has identified C. jejuni’s ability to invade intestinal epithelial cells (IECs), an important virulence trait, in vitro, but little is known regarding cell invasion in vivo, and its effect on disease progression and the immune response. Our work focuses on IEC inflammasome activation in controlling C. jejuni pathogenesis. Knockout mice lacking inflammasome components ASC-/-, Caspase-1-/-, Caspase-1/11-/-, and the cytokine IL-18-/-, were inoculated with C. jejuni 81-176. Colonic and cecal samples collected 1 and 7 days post-infection had their pathogen burdens, C. jejuni cell invasion, and histopathology assessed, along with changes in expression of the pro-inflammatory cytokines interleukin-1β and interleukin-18. Although no significant differences in luminal colonization were observed, there was a marked increase in C. jejuni present and replicating intracellularly within Caspase-1-/-and Capsase-1/11-/- IECs. In addition, wild-type mice produced significantly more IL-1β and IL-18 relative to knockouts, despite more intracellular bacteria and worsened inflammation in inflammasome-deficient mice. We propose inflammasome-mediated cleavage of Caspase-1, which releases IL-18, is responsible for combating C. jejuni invasion and replication in IECs. IL-18 induces IFN-y release by IELs to ultimately limiting C. jejuni infection through an enhanced immune response. Failure to control IEC invasion worsens downstream pathology and elevates virulence and persistence in the gut. These results highlight the importance of IEC inflammasome activation in combating C. jejuni infection and the consequences of failure to combat cell invasion. Course: Vallance Lab - Summer Student Research Program

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Group II: Immunology Session A-MSL (1:30 – 2:45pm) Authors: Leo T Liu Liposomal Formulations of Host Defense Peptides Retain Efficacy While Reducing Cytotoxicity and Aggregation. Host defense peptides are sought to be the promising alternatives to antibiotics. Their bactericidal, immunomodulatory, and anti-biofilm activities make them an attractive candidate to be for therapeutics against bacterial infections. However, there are several drawbacks of peptides that must be considered to be potentially applied in clinical settings. Here, we have attempted to improve the host defense peptide by synthesizing multiple liposomal formulations and characterizing in vitro. Successful formulations show immunomodulatory activities that host defense peptide, IDR-1018 holds, while reducing its aggregation tendency and cytotoxicity. Course: directed studies, Hancock lab

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Group II: Immunology Session B-MSL (4:00 – 5:15pm) Authors: Chris James, Ofodile Morah, Victoria Panwala, Amin Yarmand * Survival of Caenorhabditis elegans Infected with Escherichia coli DFB1655 is not Affected by a Missense Mutation in dop-1 or Treatment with Chlorpromazine Hydrochloride O antigen producing strain of E. coli MG1655, DFB1655 L9, has been shown to be pathogenic to C. elegans, with killing rates comparable to Pseudomonas aeruginosa, a known C. elegans pathogen. Inhibition of the dopamine signalling pathway has been found to increase survivability of C. elegans against P. aeruginosa infection via activation of the p38/MAPK pathway. It is not known whether loss of dopamine signalling affects E. coli infections of C. elegans. Here, we aim to investigate the effect of inhibiting dopamine signalling on survivability of nematodes against E. coli DFB1655 L9 infection. Based on the previously reported results in P. aeruginosa infection, we hypothesize that loss of dopamine signalling protects nematodes against DFB1655 infection. We used C. elegans wild type Bristol N2 strain and a dop1 mutant strain of C. elegans with a missense mutation in D-1 like dopamine receptor. We performed 10 or 12-day survival assays comparing dop1 versus WT worms against DFB1655 infection. We also used Chlorpromazine hydrochloride, CPZ, as a dopamine antagonist to inhibit dopamine signalling pathway in WT worms. Our results show that C. elegans has a significantly reduced survival percentage when growing on DFB1655 compared to growth on the parent MG1655 strain. Moreover, dop1 mutants of C. elegans showed no enhanced survivability compared to WT worms against the infection. CPZ treated WT worms also showed no increased percent survival compared to the water treated controls. This study establishes an E. coli infection model in C. elegans. Our results confirm the previous findings that O antigen expressing E. coli is pathogenic to nematodes. Moreover, our data indicates that the loss of dopamine signalling in C. elegans does not offer protection to DFB1655 infection, suggesting different host responses are elicited against E. coli DFB1655 infection compared to the previously characterized P. aeruginosa infection. Course: MICB 421

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Group II: Immunology Session A-WESB (1:30 – 2:45pm)

Author: Uyen Nguyen

The Role of Tumor Necrosis Factor Alpha (TNF) Receptor Cd120B Expression on Human Regulatory T Cell (Treg) Proliferation Using Crispr-Cas Mediated Knockout.

Tumour necrosis factor alpha (TNF) is a pro-inflammatory cytokine that is required for the

normal development of the immune system. However, TNF has also been linked to type 1 diabetes (T1D) pathogenesis. Regulatory T cells (Tregs) play a central role in maintaining self-tolerance and preventing autoimmunity development. In T1D, evidence suggests that Tregs stop working properly, leading to excessive activation of inflammatory cells and subsequent

beta cell destruction. It is known that TNF can have both positive and negative effects on Treg

function. There are two different cell surface receptors for TNF: CD120a and CD120b. Several studies have shown that CD120a stimulation induces apoptosis and CD120b promotes survival. Therefore, low CD120b expression could be deleterious for Tregs. To study the role of CD120b in human Treg function, we developed and optimized a method using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing technology to knock out (KO) CD120b expression in primary human Tregs. Then, we assessed Treg proliferation in CD120b-

KO Tregs in response to T cell receptor (TCR) stimulation with TNF. We found that while TCR-

activated CD120b+ Tregs exhibited a marked increase in proliferation in the presence of TNF,

CD120b-KO Tregs did not exhibit increased proliferation with addition of TNF. Overall, we show that CD120b plays an important role in promoting Treg proliferation, and thus could be a target for developing novel therapeutics aiming to restore immunoregulatory balance.

Course: directed studies, Levings Lab

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Group II: Immunology Session A-MSL (1:30 – 2:45pm)

Author: Jordan Brundrett

Low Affinity FcγRIIIA Promotes IVIg-Mediated IL-10 Production in Monocyte Derived Macrophages

Harvested from the blood of multiple donors, many different immunoglobulin G (IgG) antibodies are pooled to form what is known as intravenous immunoglobulin (IVIg). IVIg has been approved to treat inflammatory diseases as well as autoinflammatory diseases of the immune system, such as multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy, and lupus (24, 25). IVIg antibodies bind to a class of cell surface receptors found on macrophages known as Fcγ receptors. Different genetic variants of certain subclasses of Fcγ receptors can alter the affinity of the Fcγ receptor, which can alter one’s susceptibility to immune, infectious, and malignant diseases (20). For instance, our laboratory has shown that macrophages expressing the high affinity FcγRIIIA genetic variant have decreased capability to induce IL-10 production but inhibit the production of IL-6 and TNF pro-inflammatory cytokines in response to IVIg and LPS treatment. To elucidate this further, pro- and anti-inflammatory cytokine profiles comprising IL-10, IL-12/23p40, and TNF-α, and IL-6 were measured to determine if either the low or the high affinity FcγRIIIA affected the ability of macrophages to respond to LPS and IVIg. Macrophages with the low affinity FcγRIIIA variant produce more anti-inflammatory IL-10 than the high affinity FcγRIIIA macrophages in response to IVIg and LPS. Moreover, macrophages with the low affinity receptor variant produce more pro-inflammatory IL-12/23p40 compared to the high affinity macrophages, yet the latter produces similar levels of IL-6 and TNF relative to the low affinity ones in response to IVIg and LPS treatment. The importance of the FcγRIIIA receptor to these varying cytokine profiles of the low and high affinity FcγRIIIA macrophages was investigated further through immunoblotting where it was determined that siRNA knockdown of the FcγRIIIA receptor reduced its protein expression in the macrophages. It was determined that the low affinity FcγRIIIA receptor is required for IVIg-mediated IL-10 production and the dampening of pro-inflammatory IL-12/23p40 and TNF production upon dual IV + LPS stimulation. The high affinity FcγRIIIA receptor limits IVIg-mediated IL-10 production and the ability to control production of IL-12/23p40 and TNF cytokines. Of particular note from the results obtained was that the low affinity FcγRIIIA receptor appears to be required for the IVIg-mediated IL-10 production in order to provide a protective effect in the face of inflammatory stimuli such as LPS. These findings provide further support to the idea that FcγRIIIA genetic variations can be a risk factor for decreased IVIg responsiveness and autoinflammatory diseases such as Crohn’s and Colitis and genetic factors, in addition to others, should be taken into account by physicians when considering treatment for such conditions.

Course: directed studies, Sly Lab

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Group II: Immunology Session B-MSL (4:00 – 5:15pm) Authors: HT Le, J Atif*, DL Mara, B Castellana, J Treissman, AG Beristain. Adam8 Potentiates Trophoblast Migration by Controlling 1 Integrin-Mediated Cell Adhesion. Placental establishment in the first trimester of pregnancy is in part characterized by the differentiation of progenitor trophoblasts along the extravillous pathway. Progenitor cells committed to the extravillous trophoblast (EVT) sub-lineage acquire molecular features that promote cell migration and invasion. In particular, invasive trophoblasts express distinct cell-matrix adhesion proteins that promote binding and spreading, and produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in the control of cell motility. Among the diverse subtypes of proteases known to control cell motility, the A Disintegrin and Metalloproteinase (ADAM) family of proteases play important roles in promoting tumor cell and leukocyte migration direct cell invasion. While multiple ADAM genes are also highly expressed by invasive trophoblasts, the importance of ADAMs in controlling trophoblast migration is less well defined. In this study, the importance of ADAM8 in EVT biology is examined. Within first trimester placental tissues, in situ localization of ADAM8 was found to be specific to HLA-G- expressing trophoblasts residing within anchoring columns and decidual (uterine) tissue. Functional studies using siRNA and ectopic expression experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 controls trophoblast invasion through a protease-independent mechanism. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates 1-integrin activity and promotes cell migration through mechanism dependent on 1-integrin function. This work describes a novel non-proteolytic pro-migratory role for ADAM8 in controlling cell migration. Moreover, this study sheds insight into the importance of ADAM8 in EVT biology and placental development. Course: directed studies, Beristain Lab

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Group II: Immunology Session A-MSL (1:30 – 2:45pm) Authors: Jake Johnston*, Pierre Becquart, Jacqueline Quandt Aryl Hydrocarbon Receptor Nuclear Translocator-2 Expression Characterizes the Remyelinating and Repair potential of Oligodendrocytes in a Model of Multiple Sclerosis. MS is a chronic inflammatory and demyelinating neurodegenerative disorder characterized pathologically by vascular alterations, inflammatory infiltrates, demyelination, glial scarring, oligodendrocyte (OL) loss, and axonal degeneration. Improving myelination processes in OLs could decrease axonal loss and ultimately translate to repair and an improved prognosis for MS patients. Aryl hydrocarbon receptor nuclear transporter-2 (ARNT2) is an essential transcription factor for neuronal health. We are the first to describe its expression in glia and aim to characterize the involvement of ARNT2 in processes which regulate OPC proliferation and differentiation. We hypothesize reductions in ARNT2 may favor OPC/OL maturation and myelination. The immortalized OPC cell line oli-neu was examined before and after maturation with dibutyryl cAMP (dbcAMP). Primary neuronal-enriched cortical cultures containing OLs were derived from rat cortices at E18. Immunocytochemistry (ICC) to detect myelin proteins 2',3'-cyclic-nucleotide 3' phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) was used to assess OL maturation and ARNT2 expression. ARNT2 expression was examined in tissues from healthy mouse spinal cords and compared to experimental autoimmune encephalomyelitis, the animal model of MS, by immunohistochemistry (IHC). In healthy mice, ARNT2 was expressed by both OL and OPC (Olig2+). Increases in OLs were observed in animals at peak clinical disease. Approximately 50% of Olig2+/GFAP- cells in the healthy CNS express ARNT2, which is reduced by almost half in the grey matter at peak disease. Mature OL in mixed neuronal cultures contain CNPase+ or PDGFRï¡+ processes and express low ARNT2. Immature oli-neu exhibit elevated ARNT2 expression while ARNT2 expression is significantly reduced in mature oli-neu cells. Our studies are the first to characterize ARNT2 expression by glial populations in the healthy and diseased CNS. ARNT2 expression correlates with OPC proliferation while ARNT2 depletion is associated with cell differentiation and myelination. Our results suggest ARNT2 expression is a marker of immature or proliferating OL or OPC both in vitro and in vivo. Course: directed studies, Quandt lab

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Group II: Immunology Poster MSL 101-12P Author: Leo T Liu Bacterial Exotoxin Sequestration with Engineered Liposomes The development of antibiotic resistances is a growing problem. Indeed, even "successful" antibiotic treatment, resulting in effective bacterial lysis, often leads to intensification of general pathology due to a massive liberation of toxins. Engineered liposomes that consist of naturally-occurring lipids, namely cholesterol (Ch) and sphingomyelin (Sm), have recently been shown to function as toxin- decoys for a multitude of membrane-damaging virulence factors secreted by Gram-positive bacteria (ref). Here we show that Sm liposomes sequester virulence factors of the epidemic community- associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 and reduce necrosis in a murine cutaneous abscess model. Furthermore, we show for the first time, that liposomes bind and neutralize toxins secreted from three different strains of the potent Gram-negative pathogen Pseudomonas aeruginosa (PAO1, PA14, and hypervirulent LESB58). Course: Academic co-op, Hancock Lab

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Group II: Immunology Poster MSL 101-14P Author: Brayden M. Wilkinson The Role of Macrophages and Neutrophils During -cell Development and Regeneration Post-injury in Zebrafish. Macrophages are phagocytic cells that are primarily involved in innate immunological responses, but increasing evidence has linked them to developmental and metabolic processes as well. Additionally, macrophages have been shown to play a critical role in facilitating the proper development of β-cells in mice. However, it has yet to be determined whether or not macrophages are involved in the regeneration of zebrafish β-cells after complete depletion – a process that occurs in zebrafish. As well, the role of neutrophils in β-cell development and regeneration have yet to be clarified. In this project, the roles of these two innate immune cell types during development and regeneration (post-injury) will be explored through the use of transgenic zebrafish β-cell/macrophage and β-cell/neutrophil ablation models. Between 2-9 dpf, it was shown that neither macrophages nor neutrophils appear within a functionally relevant distance of the beta cells, but rather, in laterally adjacent tissue. Additionally, it was shown that neither macrophages nor neutrophils accumulate within the islet for an extended duration (>24 hr) during induced β-cell ablation (3-5 dpf) or during the regeneration process (5-9 dpf). Genetic ablation of neutrophils resulted in no detectable impairment either to the developmental proliferation of β-cells from 4 dpf onwards nor to the regeneration of genetically ablated β-cells. Although the genetic ablation of macrophages similarly had no detectable impact on the development proliferation of β-cells from 4 dpf onwards, macrophage-depleted zebrafish exhibited complete impairment to regeneration. Course: directed studies, Kieffer Lab

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Group II: Immunology Poster MSL 101-18P Authors: Yi Tian Liu*, May Dang-Lawson, and Michael R. Gold Inhibition of LIM Kinase (LIMK) Impairs B Cell Receptor-Induced B Cell Spreading By Causing Hyperactivation of the Actin-Severing Protein Cofilin B cells are important components of the adaptive immune system, providing humoral immunity by producing antibodies that neutralize and opsonize pathogens. Foreign antigens presented on the surface of antigen-presenting cells (APCs) can activate B cells by binding to the B Cell Receptor (BCR). This activation process requires extensive remodeling of the actin cytoskeleton, which allows the B cell to spread on the surface of the APC in order to scan for antigen and form an immune synapse (IS). Activation of cofilin, an actin severing protein, is essential for BCR-induced remodeling of actin, B cell spreading, and IS formation. In this study, we investigated the effects of inhibiting LIM domain kinase (LIMK), a negative regulator of cofilin activation, on BCR-induced activation of cofilin and B cell spreading. We hypothesized that inhibition of LIMK would lead to an increase in the amount of active dephosphorylated form of cofilin, as well as increased B cell spreading. We found that treating A20 B-lymphoma cells with LIM Kinase Inhibitor (LIMKi3) resulted in enhanced BCR-induced de-phosphorylation/activation of cofilin. However, contrary to our hypothesis, this was associated with decreased B cell spreading on immobilized anti-Ig antibodies and a much thinner peripheral ring of F-actin. This is consistent with a model in which the severing of actin filaments by activated cofilin has two functions, providing actin monomers for increased actin polymerization at the cell periphery, and disassembling actin at the inner face of the peripheral actin ring in order to promote cell spreading. Our findings suggest that LIMK, presumably via its effects on cofilin, is a key regulator BCR-induced of actin remodeling that controls B cell spreading. Course: directed studies, Gold Lab

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Group Immunology Poster MSL 101-10P Author: Wallace Yuen The Role of STAT1 in Coordinating CD8+ T cell-mediated Responses to Murine Norovirus Murine Norovirus (MNV) is an enteric virus and the CR6 strain persists in wild-type hosts in the absence of clinical histological signs. However, in hosts lacking STAT1, persistent MNV CR6 infections cannot be controlled and can results in lethal outcomes. STAT1 is an important transcription factor in interferon signaling and mediates an antiviral state in the host cell. The exact mechanism for why STAT1-deficient hosts cannot control persistent MNV infection is poorly studied, but our lab has established that this is associated with an expansion of antiviral CD8+ T cells in Stat1-/- (Stat1KO) mice compared to littermate control Stat1+/- (Stat1Het) mice. Here, I tested the hypothesis that Stat1Het bone marrow-derived dendritic cells (BMDCs) are haplosufficient compared to Stat1WT BMDCs. Then, through the use of an adoptive T cell transfer design, I demonstrate that STAT1 appears to be a partial CD8+ T cell-intrinsic requirement in controlling persistent CR6 infections. This is in contrast to an acute MNV CW3 infection, where the presence of STAT1 is required for CD8+ T cells to control viral infection. Together, this data provides preliminary support that Stat1Het mice may be used instead of Stat1WT mice and suggests that STAT1 signaling may be a cell-intrinsic requirement in priming CD8+ T cells in MNV infections. Course: directed studies, Osborne Lab

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Group III: New Concepts in Virology Session B-WESB (4:00 – 5:15pm) Authors: Niknaz Malekafzali Biological Individuality: The Diversity of Intestinal Virome Onsets Type I Diabetes in Genetically Susceptible Children. Type 1 diabetes (T1D) is a chronic multifactorial autoimmune disease that affects about 20 million individuals worldwide. The etiology of T1D is unknown, however, it is believed that a strong genetic component interacts with environmental factors leading to the onset of this disease. The intestinal microbiota is an environmental risk factor that has been implicated in the pathogenesis of T1D, however, the exact mechanism by which intestinal microbiota contributes to the development of this disease is unclear. Intestinal microbiota is unique to each individual and the composition of this community has been identified to play a major role in human’s health and diseases as it is involved in the development and homeostasis of the immune system. Therefore, it plays an important role in each individual’s response to infectious agents and development of diseases. Although many studies have focused on the association of intestinal bacteriome and type 1 diabetes, the research on the association between intestinal virome and development of T1D remains limited in number. Since studies have shown differences in the diversity and abundance of intestinal virome in T1D patients, the intestinal virome could serve as a potential answer for the unknown etiology of T1D. Therefore, analysis of the changes in the intestinal virome before seroconversion of T1D-associated autoantibodies would open doors for the development of novel diagnostic tools, preventative measures, and therapeutic avenues including possible personalized treatments. Therefore, this article provides evidence supporting the association between the intestinal virome and type 1 diabetes, an overview of the biology and biological significance of Circoviridae family that has be identified to possibly offer protection from development of T1D and the proposed mechanism for how a shift in the intestinal virome leads to onset of T1D. Course: MICB 406

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Group III: New Concepts in Virology Session B-WESB (4:00 – 5:15pm) Authors: Andrew P. Golin* A Cure for Cancer? The Therapeutic Potential of Combining Oncolytic Viruses and Chimeric Antigenic Receptor T-Cell Therapy. Cancer continues to be a devastating diagnosis worldwide. With an aging population and thus an anticipated increase of cancer incidence, it is critical that discovery, testing, and refinement of novel treatments continue. Fortunately, active research has developed and improved two cancer therapies: oncolytic virotherapy and the novel chimeric antigen receptor (CAR) T-cell therapy. The former utilizes oncolytic viruses (OVs) that preferentially infect and kill cancer cells over healthy, non-cancerous tissues. The latter isolates the patient’s T-cells, uses a disarmed virus to insert genes expressing genetically engineered receptors, known as chimeric antigen receptors, that target tumor-associated antigens on malignant cells. Despite several clinical successes with both therapies, limitations exist which prevent their medicinal potentials from being achieved. However, it has been previously reported that the administration of oncolytic viruses into patients, even with usual sequestration levels, can result in the release of neoantigens from tumors which may promote the often-poor entry of CAR T-cells into solid tumors. Furthermore, oncolytic viruses have shown to reverse the immunosuppressive environment of certain tumors. With this change of immunogenicity, CAR T-cells may then be able to perform its primary objective of orchestrating the immune system to kill malignancies. This project seeks to investigate the current limitations of oncolytic virotherapy and CAR T-cell therapy, the therapeutic synergy when combining OVs with CAR T-cells, and the risks of this novel combinatory therapy. For the millions of individuals who currently have and will develop refractory cancers, the need for an effective cancer therapy cannot be overstated. Research in oncology therefore cannot not be limited to undiscovered treatments but must also investigate the effects of combining currently available treatments. Course: MICB 406

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Group III: New Concepts in Virology Session B-WESB (4:00 – 5:15pm) Authors: Syed S Raza* Salivary Exosomal microRNAs: Discovery and Potential Biomarkers for Human Papillomavirus- Associated Oral Squamous Cell Carcinoma. Exosomes are small, membrane bound vesicles secreted by many types of cancers including oral squamous cell carcinoma (OSCC), and have been found to contain various cargo, including microRNAs (miRNAs). miRNAs are short, single-stranded non-coding RNAs that can post-transcriptionally regulate gene expression. OSCC, the sixth most common cancer in the world, has a dismal survival rate of only 50% over five years. This is in part due to the late diagnosis of the cancer as it is very difficult to identify a lesion at risk during the earliest stages of the disease as well as its high recurrence rate. The goal of this study is to identify and exploit the presence of exosomal miRNAs secreted in bodily fluids such as saliva to diagnose early stages of OSCC. The development of a technique to detect the presence of this disease during its early onset will help provide timely and effective treatment to patients all around the world and thus increase the overall survival rate. In this paper, we discuss whether infection with HPV results in the release of specific miRNA biomarkers in exosomes and if these miRNAs of interest are able to be detected in saliva during the early stages of the cancer. We also explore the possibility of detecting other HPV-associated cancers using these miRNAs in order to streamline the cancer screening process and to detect the presence and progression of multiple cancers in a non-invasive manner using only one or two types of bodily fluids as samples. This field of ‘liquid biopsies’, where bodily fluid samples are examined for biomarkers to identify the presence of a disease is quickly becoming the favoured approach to diagnosing many different health complications, especially cancer, and has a lot of potential in the future. Identification of potential salivary microRNA biomarkers for HPV-associated OSCC will revolutionize cancer diagnostics and allow patients throughout the world to have increased life expectancy. Course: MICB 406

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Group III: New Concepts in Virology Session B-MSL (4:00 – 5:15pm) Author: Boyuan Zheng A Double-Edged Sword: The Therapeutic Potential of Chimeric Antigen Receptor T Cell in HBV/HIV Co-Infected Patients. Hepatitis B virus (HBV) is a double-stranded DNA which can cause life-threatening chronic liver disease. HBV is currently one of the leading cause of human death. Approximately 350 million people worldwide are chronically infected with HBV, and up to a quarter of this infected population is at risk of death from cirrhosis, liver failure, or hepatocellular carcinoma. Coinfection with human immunodeficiency virus (HIV) is common in people who inject drugs (PWID). The HBV/HIV coinfection increases both the morbidity and mortality rates. This represents a significant global health burden. Although many current HBV antivirals have a dual effect in treating HIV infection and are able to control viral replication, they are insufficient to eradicate the virus. Chimeric antigen receptor (CAR) T cell immunotherapy has great potential in targeting infected hepatocytes presenting HBV surface antigens (HBsAg) as means to eliminate HBV infection. The adoptively transferred immunity may be beneficial in HIV patients as CAR T cell may facilitate both cellular and humoral immune response. Challenges of such therapy may include transient liver damage as a side effect, as well as some economic burdens. Multiple critical factors need to be considered to develop a successful CAR T cell therapy. Despite the current obstacles, research focus towards CAR T cell therapy may elucidate substantial potentials in treating not only chronic HBV infections, but also chronic HCV infection and HIV coinfection. Course: MICB 406

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Group III: New Concepts in Virology Session A-WESB (1:30 – 2:45pm) Author: Felix Ma Discovery and Characterization of Blood-Brain Barrier Targets to Develop New Therapeutic Strategies to Treat Neurotropic Viral Infections. There has been a rising global health threat of emerging and re-emerging neurotropic viruses. High morbidity rates in combination with expanding vector populations have led to greater populations of people at increased risk. Neurological viral infections can manifest as encephalitis and/or meningitis with potential for brain injury and disability, causing a financial impact in addition to the significant health impact. It is known that neurotropic viruses can enter the central nervous system (CNS), but the mechanisms by which these viruses can actually cross the blood-brain barrier (BBB) are not clear. There is a pressing need to develop antiviral treatments as treatment options are lacking for neurotropic viruses. With a focus on neurotropic arboviral flaviviruses, viral molecular determinants for BBB crossing were determined. Then, host receptors and pathways that are hijacked by neurotropic viruses for BBB crossing were identified. The gaps in understanding the mechanism of BBB crossing can begin to be closed by determining the key players of the mechanism, although direct roles may not be known. Lastly, these identified viral and host factors were applied to propose several direct-acting and indirect-acting antiviral drug candidates that may be effective at treating neurological viral infection. There is a potential for combining the proposed treatments and development of a multidrug regimen that provides more complete prevention against CNS entry by neurotropic viruses. These proposed antiviral treatments may address a real need for a potential impending outbreak of neurotropic viruses. Future directions beyond the antiviral development may include development of much-needed neurotropic viral diagnostic tests, as well as to develop an effective drug delivery system to the CNS for application of antiviral treatments against neurological viral infection. Course: MICB 406

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Group III: New Concepts in Virology Poster MSL 101-15P Author: Judy Ban* Targeted Delivery of Therapeutic Exosome to HIV-1 Reservoir Cells: A New Strategy to HIV-1 Eradication in Children. The human immunodeficiency virus type 1 (HIV-1) is a retrovirus that permanently integrates into the host genome of CD4 positive T cells. The virus can drastically increase the risk of opportunistic infections and tumour-development. Although the invention of the highly active antiretroviral therapy (HAART) has dramatically reduced morbidity against AIDS-associated malignancies, over 77% of HIV-positive children in Sub-Saharan Africa still lack access to the treatment today. Without treatment, more than 50% of them will die by 2 years of age due to rapid disease progression to serious immunodeficiency. With emerging understanding of extracellular vesicles in intercellular communication, clinical applications of exosomes may answer the growing demand for more targeted, holistic and convenient HIV diagnostics and therapeutics in resource-poor countries. This review will investigate the potential applications of exosomes from three perspectives. First, exploration of the underlying mechanisms of HIV persistence and locations of HIV reservoirs in children. Second, we will propose ways to target engineered exosomes to these reservoirs, and how to select the most appropriate therapeutic tool as exosome cargo. Last, to follow up on the treatment progress and disease progression, the potential development of an exosome-based liquid biopsy for omic-profiling will be examined. A review of existing literature indicated the location of HIV-1 reservoirs in children to be resting T cells, memory T cells, T regulatory, Th17 and Th2 cells. We propose to engineer the surface of therapeutic exosomes with HIV-1 envelop proteins and host tetraspanin for specific targeting to reservoir cells, and deliver micro-RNA and gene editing tools for the elimination of the virus from host cells. Isolation of host-produced exosomes and omic-profiling of exosome cargo can be performed for a holistic view of HIV-1 disease progression and treatment effects. The success of exosome-based HIV therapy has the potential to help overcome the burden of maintaining continuous accessibility and resources to supplement the life-long dependency on HARRT, for children and adults in Sub-Saharan Africa. Course: MICB 406

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Group III: New Concepts in Virology Poster MSL 101-16P Author: Leilynaz Malekafzali* Emerging Role of Circulating MicroRNAs and Opioid-mediated Enhancement of HIV-1 Infection in People Who Inject Drugs (PWID): Opportunities for Biomarkers and Novel Antiviral Strategies. HIV is a global pandemic with 36.7 million people living with HIV/AIDS worldwide in 2016. There is no cure for HIV and all the current treatments control the active viral replication. The risk of HIV infection is 59 times higher in people who inject drugs (PWID) compared to the rest of the population due to the syringe sharing. Opioids are used for chronic pain management but due to their widespread use they have been associated with drug abuse, overdose and death. The addiction to opioids in PWID increases their susceptibility to HIV-1 infection through needle sharing. Additionally, recently some researches are showing that opioids further increase the susceptibility of individuals to HIV-1 infection by the down-regulation of host coded anti-HIV microRNAs. However, the mechanism by which opioid causes the depletion of the anti-HIV miRNAs is unknown. The observation of the up-regulation of the anti-HIV miRNAs associated with exosomes in plasma after the opioid treatment, suggests a link between the opioid treatment and induction of the exosomal release of the cellular anti-HIV miRNAs. Anti-HIV miRNAs modulate HIV-1 infection and replication either by targeting HIV genome or by targeting the host cellular mRNAs that code for the proteins required for the successful replication and entry of the virus. The systemic delivery of liposomes with packaged anti-HIV miRNAs targeted to the cellular reservoirs of HIV-1 is a proposed preventive mechanism for opioid addicts and a therapeutic tool for the HIV-1 infected individuals who were/are addicted to opioids. This article explores the association between opioids and the differential expression of anti-HIV miRNAs, the impact of anti-HIV differential expression on the susceptibility of individuals to HIV-1 infection, and the potential preventive and therapeutic options for HIV infected individuals who are addicted to opioid and/or using opioid agonists. Understanding the impact of opioids on the susceptibility of individuals to HIV-1 infection helps to educate the population, prevent the new incidents of the disease, and further inform us of the new preventive and therapeutic tools. Course: MICB 406

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Group III: New Concepts in Virology Session B-MSL (4:00 – 5:15pm) Author: Sami Rahman MERS-CoV: Identifying Viral and Host Factors Associated with Transmission, Pathogenesis, and Treatment Development. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel betacoronavirus that is associated with a high mortality rate (~36%) and is endemic in the Middle East. Since 2012, there has been 2100 laboratory confirmed cases and 730 deaths associated with MERS-CoV in 27 countries. Clinical manifestations of the virus range from asymptomatic to acute respiratory distress syndrome (ARDS), myocarditis, septic shock, and renal failure resulting in death. The severity of disease and fatality rate is much higher in patients with existing co-morbidities such as diabetes and hypertension. Bats are the natural reservoirs of MERS-CoV and can travel over long distances to transmit the virus to different intermediate reservoirs and humans. This factor combined with the ability of the MERS-CoV S protein to undergo mutations to increase its transmission efficiency gives the virus a pandemic potential. Currently, there is a lack of knowledge on how viral and host factors contribute to the transmission and pathogenesis of MERS-CoV in humans. This article explores the different viral and host factors associated with MERS-CoV transmission and pathogenesis, and how this knowledge can aid in the development of therapeutics. MERS-CoV transmission is facilitated by interaction between the viral receptor binding domain of S1 and host receptor dipeptidyl peptidase 4 (DPP4). MERS-CoV can utilize multiple host proteases such as furin, transmembrane serine proteinase 2 (TMPRSS2), trypsin, and cathepsin to cleave the S protein, which facilitates membrane fusion. MERS-CoV has been shown to primarily infect cells in the lower respiratory tract of humans, but it can also infect immune and kidney cells. An overactive inflammatory response, induction of apoptosis, and cytokine dysregulation are vital factors that contribute to the high pathogenicity associated with MERS-CoV infection. Future treatment options for MERS-CoV include inhibition of virus entry, fusion, polyprotein cleavage, and replication by targeting both viral and host factors. Course: MICB 406

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Group III: New Concepts in Virology Session A-WESB (1:30 – 2:45pm) Author: Maria-Elizabeth Baeva* Crossing barriers: How John Cunningham virus crosses the blood brain barrier and induces progressive multifocal leukoencephalopathy in patients on disease-modifying therapies. Progressive multifocal leukoencephalopathy is a fatal demyelinating disease caused by the John Cunningham virus (JCV). While it is known to cause lytic infection in oligodendrocytes, many questions remain about this seemingly asymptomatic and dormant virus that infects approximately 90% of the population during childhood. JCV becomes neurotropic under very specific conditions such as autoimmune disease and the use of certain disease-modifying therapies (DMTs), specifically rituximab and natalizumab. This paper addresses questions about how JCV crosses the blood brain barrier, what genetics from the host and the virus enable neurotropism and virulence and how monoclonal antibody drugs aid in the reactivation of the virus. Based on the current literature, the rearrangement of the non-coding control region and VP1 capsid protein are key determinants of whether the virus becomes neurotropic. Host immune factors such as permissive alleles and overexpression of Spi-B can also lead to immune evasion and increased viral replication by JCV. A model is proposed wherein JCV use B cells as a ‘Trojan Horse’ to cross the blood brain barrier. Co-infection with Epstein-Barr virus (EBV) can result in B cell fusion with oligodendrocytes and transmission of JCV. Finally, disease-modifying therapies play a role in activating JCV by primarily suppressing the immune system and upregulating the migration of leukocytes, allowing the mobilization of JCV-infected hematopoietic stem cells and/or B cells to the blood brain barrier. Future studies should be done to conclusively determine the roles that Spi-B, MHCI molecules, hematopoietic stem cells and EBV co-infection play in transmission, pathogenesis, and genetic rearrangement of JCV. The goal is to one day create an anti-viral or vaccine against JCV so patients can continue to be treated with effective DMTs without the threat of a fatal adverse reaction. Course: MICB 406

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Group III: New Concepts in Virology Session B-MSL (4:00 – 5:15pm) Author: Shivani Mysuria Leishmania RNA Virus: Mere Endosymbiont or Major Player in Leishmania-associated Disease? Leishmaniasis is caused by Leishmania protozoan parasites which are transmitted by the bites of infected sand flies. Approximately 12 million are currently infected with the disease, and there are over 1 million new cases every year. Current treatments involve chemotherapy and medications against the parasite, but are associated with adverse effects and drug resistance. Without effective treatments, individuals can be left with debilitating physical deformities, and can lead to death if left untreated. Some parasite strains have been found to be infected with Leishmania RNA virus (LRV). LRV contains a dsRNA genome that encodes a capsid protein and RNA-dependent RNA polymerase, and resides exclusively in the cytoplasm of the parasite. Remarkably, LRV has been implicated with increased rates of disease relapse, drug treatment failure, and exacerbated disease symptoms. Investigating the role LRV plays in Leishmania-associated disease may open up novel therapeutic avenues that can better treat Leishmaniasis and prevent future infections. An overview of the biological relationship between LRV and Leishmania parasites, what effects LRV has on Leishmania-associated disease, and whether the virus can be specifically targeted as a therapeutic strategy will be explored. Understanding LRV’s role and contribution to disease pathogenesis will increase our understanding of virally-infected parasites. Course: MICB 406

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Group III: New Concepts in Virology Session A-MSL (1:30 – 2:45pm) Author: Priya Suresh Zika Virus Infection: A Cause or Explanation in the Development of Guillain–Barré Syndrome? Guillain–Barré syndrome (GBS) is an autoimmune disease that damages the nerves of the peripheral nervous system, causing paralysis in humans. Several infectious agents including dengue virus, Epstein-Barr virus, and herpes virus have been suggested to be possible causes in the development of GBS however the exact cause is yet to be discovered. Recently a strong temporal and geographical association between the Zika virus (ZIKV) outbreaks and an increase in GBS cases within the region has implied that ZIKV could possibly portray a key role in the onset of GBS. Large numbers of infected individuals have been reporting of neurological complications, such as microcephaly, following ZIKV outbreaks. The Asian strain of the ZIKV in particular, has been shown to be neurovirulent and therefore capable of infecting nerve cells providing further support for this connection. This article discusses the current evidence available to support the association between ZIKV and GBS, the molecular determinants that increases the neurotropism of specific ZIKV strains, and the proposed molecular mechanism of ZIKV pathogenesis that causes the development of GBS. Determining the role of ZIKV in the onset of GBS can allow for the discovery of novel diagnostic biomarkers, which can aid in the early detection and prevention of GBS. The discovery of a possible mechanism of neurovirulent ZIKV strains can aid in understanding the pathogenesis of the virus in not only GBS, but other associated neurological diseases as well. Course: MICB 406

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Group III: New Concepts in Virology Poster MSL 101-17P Author: Haya Abuzuluf Human Endogenous Retroviruses as Contributors of Exosome-mediated Autoimmunity in Systemic Lupus Erythematosus. At least 8% of the human genome was formed by integration of retroviral DNA sequences. These sequences are remnants of ancient infections that have accumulated mutations over millions of years. Although human endogenous retroviruses (HERVs) are previously thought to be inactive, they are now emerging as potential contributors of autoimmune diseases. Environmental and epigenetic factors have been shown to activate the expression of HERVs in pathological conditions such as Systemic Lupus Erythematosus (SLE). SLE is a poorly understood non-organ specific autoimmune disease lacking diagnostic criteria and curative treatments. It has been suggested that an unfortunate interplay of genetic susceptibility and environmental factors play an important role in generating an abnormal autoimmune response in SLE patients. HERVs have been suggested as the bridge linking environmental factors to the pathogenesis of SLE. No clear mechanisms of causation have yet been identified in this association. This review outlines the key HERVs implicated in SLE with factors influencing their activation, the proposed mechanisms by which HERVs are predicted to utilize in SLE etiopathogenesis and the potential of HERVs in contributing to exosome-mediated autoimmunity in SLE. I propose an experimental approach where isolated exosomes from lab confirmed SLE cases are investigated through deep sequencing and tandem mass spectrometry to establish full proteomic and transcriptomic profiles. This will allow the characterization of exosomal cargo to identify HERV products or sequences that may be transmitting intercellular signals involved in the immune dysregulation of SLE. Elucidating the association between HERVs and SLE may facilitate the development of novel diagnostic and therapeutic tools to combat this debilitating multi-system disease. Course: MICB 406