2016 Biomedical & Biological Sciences & Beyond Symposium, Lecture notes of Microbiology

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2016 Biomedical & Biological

Sciences & Beyond

Symposium

th

Annual

Connect. Collaborate. Create.

2016 Symposium Organizing Committee Gwendolyn Beacham (^) Ezen Choo Erin Chu Shing Hu (^) Jordan Mohr Erin Nicklow Kristeen Pareja (^) Jocelyn Wang (^) Julio Sanchez Kevin Siegenthaler Aravind^ Sivakumar Divya Shiroor

Arla Hourigan Faculty Advisors: Chairs: Frances Chen Martin Ian Malgapo Office of Graduate Education: Gunther Hollopeter (^) Holger Sondermann Cindy Grey 2016 Symposium Organizing Committee

The Research Lecture The annual BBS Symposium’s Keynote Address is named for the Baker Institute’s own Dr. Douglas D. McGregor. Dr. McGregor served as Director of the Baker Institute from 1976 - 1991 , and subsequently served as Associate Dean for Research and Graduate Education for ten years. Dr. McGregor received his M.D. from the University of Western Ontario and earned his D. Phil. from the University of Oxford. He was awarded an honorary Doctor of Veterinary Science Degree from the University of Sydney in 2007 , and in 2012 was named Honorary Associate of the Royal College of Veterinary Surgeons. During an active research career that spanned four decades, Dr. McGregor conducted seminal research on lymphocyte function and the early immune response, making immeasurable contributions to the field of immunology. His substantial contributions to veterinary research and education at Cornell include securing funding for the NIH Comparative Medicine Training Grant and creating the Leadership Program for veterinary students, which provides an intensive, research-oriented summer experience for veterinary students who seek to broadly influence the veterinary profession through a science-based career.

Faculty Speakers: Dr. Pamela Chang Microbiology and Immunology Regulation of macrophages by gut microbial metabolites Dr. Richard Cerione Molecular Medicine How Rho GTPases led us to new areas of biology and disease Dr. Gunther Hollopeter Molecular Medicine Molecular mechanisms of endocytosis revealed by genetics

Broadening Experiences in Scientific Training a Cornell BEST Program: Rethinking PhD Training A new paradigm for PhD education FASEB 30(2016) Trained scientists are needed in many sectors of our economy and in academia. The BEST (Broadening Experiences in Scientific Training) program provides accurate career information and career-oriented experiences to graduate students and post-docs. The goals are to help trainees make informed choices about their career and to help them find career matches that best fit their individual skills and strengths. With these goals in mind the nationwide consortium of 17 institutions awarded BEST grants funded by the National Institutes of Health are working together to redefine the paradigm for PhD education. See http://www.fasebj.org/content/ 30 / 2 / 507 .long The Cornell BEST Program provides detailed information to STEM trainees about career options as well as active learning customized to specific career tracks in Science Policy; Industry, Entrepreneurship & Management; Science Communication; and Governance, Risk & Compliance. Getting involved in BEST requires a short application, but everyone is different and the BEST experience can include individual consultations with career mentors, participation in seminars, hands-on workshops, involvement in clubs, taking courses, site visits, and/or trainee initiated projects. Flexible “BESTernships” with advisor consent are also encouraged. Participation is voluntary according to individual time constraints, but the more you invest—in planning, exploring, and experiencing in order to make informed career decisions— the more you will benefit. Numerous fruitful outcomes have emanated from trainees’ participation in BEST, which have led to further successes in science policy, science communication, industry and entrepreneurship, including company formation and securing federal Small Business Innovation Research (SBIR) funding.

Cornell BEST Program Senior Director: Susi Varvayanis Communication (^) Science Policy Governance, Risk & Compliance Industry, Entrepreneurship & Management Mentoring Cornell BEST trainees have gone on to advocate for and practice what they learn. Through mentorship they have learned about the writing and editing process. They have authored articles in the BioMed Breakthroughs Industry Report, the Atlanta BEST Magazine; run and won business case competitions; interviewed for alumni magazines; and shared their opinions on energy generation, and even the future of the postdoctoral experience with Science. They have also given keynote talks, formal presentations at industry conferences, participated in live Science Cabarets and won prestigious policy fellowships. Several trainee-run clubs have been formed including the Cornell Graduate Consulting Club (CGCC), Advancing Science and Policy (ASAP); and others collaborate with the BEST Program, such as the Technology & Entrepreneurship Club (TEC) and Biotechnology Club. Together they provide training, interactions with practitioners in the field, case competitions, practice describing their expertise in the language of their future employer (or funder), and practical advice on how to find and land a job using the skills learned. Another successful outcome is that trainees might increase their resolve to become tenure track academics, but with increased skills and as a consequence of an informed decision. (^1) http://www.fasebj.org/content/30/2/507.long (^2) http://best.cornell.edu/index.cfm/news.details?newsID= (^3) http://best.cornell.edu/index.cfm/page/about/FAQ.htm As any ‘BESTie’ will tell you, the more you engage, the more you benefit. This and many other lessons, like ‘opportunities aren’t found, they’re made’^2 , underscore the importance of honing skills beyond your technical expertise for your future success.

Oral Presentations 1 Combinatorial Adhesive Codes In Single Neurons Adam Bisogni^1 , Shila Ghazanfar^2 , Jean Yang^2 , and David Lin^1 (^1) Department of Biomedical Science, Cornell University, Ithaca, NY (^2) Department of Mathematics and Statistics, University of Sydney, Australia The human brain consists of approximately 86 billion neurons, which form an estimated 1 x 10^16 synapses. A long standing model hypothesizes that neurons use combinatorial codes of axon guidance cues to generate a large amount of cellular diversity with only a small number of genes. Such diversity endows neurons with the ability to find and connect with their proper targets, while avoiding incorrect ones. To determine the combinatorial code used by neurons, we employ a novel single cell RNA analysis method to define the patterns of axon guidance cues expressed in individual neurons. We focus on the protocadherins, a cell adhesion gene family known to influence axon guidance. Using this approach, we show that in single neurons, the protocadherins are expressed in a wide range of combinations, confirming their ability to generate cell surface diversity. We then utilize functional adhesion assays to show that combinations of protocadherins can contribute critical roles in neuronal recognition. Together, these studies define combinatorial codes of protocadherins within single neurons, and demonstrate the functional significance of the combinatorial code. 2 Maternal exposure to high-fat diet increases sweet taste response and sweet taste receptor mRNA expression in adult offspring Ezen Choo^1 and Robin Dando^2 (^1) Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY (^2) Food Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY Based on epidemiological and animal studies, a maternal high fat (HF) diet predicts offspring preference for fatty foods. Additionally, maternal body mass index and gestational weight gain predict future over-weight/ obese status in children and adolescents. In rodent studies, maternal junk food consumption during pregnancy produces offspring that over consume and prefer junk food, suggesting an increased preference for foods rich in fat, sugar, and salt. Whether the underlying basis for this is due to changes in taste perception remain to be investigated. In this study, dams were fed a control or HF diet before and during pregnancy and then all offspring were maintained on control diet after weaning; thus, the only experience with HF food for the offspring was through maternal exposure during early development. Taste change was assessed in adult offspring using brief- access taste testing. Offspring of HF fed dams showed increased sensitivity to sucrose (F2,255=7.130, p=0.0010). We hypothesize that this increase in sensitivity results from changes in the expression profile of taste buds for sweet taste receptors. We performed qRT-PCR to assay taste receptor expression, and found that both subunits composing the sweet receptor heterodimer to be increased in the offspring of HF fed dams (T1R2 p=0.0001, T1R3 p=0.0033). The results indicate that the taste behavior changes in the adult offspring induced by maternal HF diet exposure correlate with increased expression of sweet taste receptors in the taste buds. We hypothesize that sweet taste receptor expression is modulated in the offspring through epigenetic regulation prior to weaning.

3 Extracellular substrate stiffness regulates macrophage response to foreign DNA Erika Gruber, Siddhartha Sinha, and Cynthia Leifer Department of Microbiology & Immunology, Cornell University, Ithaca, NY Macrophages are important in innate immunity, tissue repair, and tissue homeostasis, and can differentiate into a wide variety of functional phenotypes in response to biochemical stimuli, such as cytokines. Recent work has shown that biophysical stimuli, such as extracellular substrate stiffness, can mediate differentiation and function of multiple cell types through a process known as mechanotransduction. Less is known about how macrophages and other immune cells respond to biophysical cues and transmit mechanotransduction signals. To investigate this, we cultured murine macrophages on tunable polyacrylamide gels that approximate different tissue stiffnesses. Macrophages on rigid surfaces were more adherent and had a larger surface area and reduced circularity compared to macrophages on compliant gels. Interestingly, macrophages on rigid surfaces secreted less tumor necrosis factor, [TNF], a pro-inflammatory cytokine, in response to CpG DNA stimulation of Toll-like receptor 9 (TLR9). The attenuated response was not due to decreased uptake of CpG DNA, which implicates altered expression, trafficking, and/or signaling of TLR9 itself. Targeted inhibition of key mechanotransduction proteins has revealed a role for classic mechanotransduction signaling (e.g. integrin-mediated activation of focal adhesion kinase [FAK] and rho-associated coiled-coil forming kinase 1[ROCK1]) and a more recently described mechanosensor, transient receptor potential vanilloid 4 (TRPV4). These studies demonstrate that the mechanical properties of the extracellular environment and mechanotransduction have a direct impact on macrophage function. Understanding the roles of mechanotransduction in regulating macrophage function could have important implications in a variety of pathologic conditions that involve macrophages and altered tissue stiffness such as fibrosis, atherosclerosis, and neoplasia. 4 Raltegravir exhibits antiviral, anti-inflammatory, and anti-angiogenic effects during feline herpesvirus (FHV1) infection. Matthew R. Pennington , Lauren Tofano, Jennifer Grenier, and Gerlinde R. Van de Walle Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY Feline herpesvirus type 1 (FHV1) is the most common viral cause of ocular surface disease in cats, frequently leading to blindness. In order to identify antivirals against FHV1, we recently showed that the retroviral integrase inhibitor raltegravir was capable of inhibiting FHV1 in cell culture as well as in a corneal explant model. We then adopted a dual RNA sequencing approach to study the effects of raltegravir therapy on both host and viral gene transcription. We initially used this data to investigate the antiviral mechanisms of raltegravir. Raltegravir therapy has been described to target either viral DNA replication (e.g. herpes simplex virus type 1) or DNA packaging (e.g. cytomegalovirus), depending on the herpesvirus. We found that with raltegravir treatment, 3 immediate early genes were upregulated, no change was observed in early gene expression, and 10 late genes were down regulated. This pattern of gene expression suggested that raltegravir inhibits FHV1 DNA replication. However, we observed (i) a more significant reduction in extracellular produced virus than in viral DNA genome copies following raltegravir treatment and (ii) a different gene expression pattern following phosphonoacetic acid treatment, a known inhibitor of DNA replication. These results collectively indicate that DNA replication might not be the major target of raltegravir, but that packaging might also be affected. Studies investigating whether raltegravir indeed inhibits DNA packaging are currently ongoing. Furthermore, we observed that raltegravir led to an upregulation of host anti-angiogenic factors during FHV1 infection. Finally, we observed that raltegravir led to an upregulation of a number of anti-inflammatory factors, including heme oxygenase 1, and downregulation of pro-inflammatory factors, such as hyaluronan synthase 2, in uninfected cells. Raltegravir- induced anti-angiogenic and anti-inflammatory effects could be beneficial during FHV1 ocular infection, as angiogenesis and inflammation are major contributors of corneal damage.

7 The human lung is a site of HIV replication during long term ART treatment: novel tools to study tissue HIV reservoirs David Gludish and David Russell Field of Comparative Biomedical Sciences, Cornell University, Ithaca, NY Background: We have developed novel tools to probe HIV biology in potential tissue reservoirs that persist during ART (anti-retroviral treatment). Our previous work found HIV mRNA in alveolar macrophages (AMs) of HIV patients. However, current methods to demonstrate such reservoirs require expensive tools such as the quantitative viral outgrowth assay (QVOA) and rely on bulk population PCR assays that lack cellular resolution. To determine if AMs produce infectious HIV, we sought robust, rapid readouts amenable to flow cytometry at point of care. Methods: We have built novel tools to identify infected HIV cells in vivo. We generated reporter cells that express GFP dependent on HIV Tat and Rev protein (TZM-GFP). To identify HIV within single cells, we developed a fluorescent in situ hybridization assay that is quantified by flow cytometry. We sorted fixed HIV-positive cells from experimental cultures and from the blood and bronchoalveolar lavage of Malawian HIV-infected adults and perform RNAseq profiling in vivo, an experiment not previously possible. Finally to purify live HIV-infected cells from patient samples, we developed a synthetic reporter RNA that expresses mCherry in HIV-infected cells using straightforward and scalable methods. Results: Clear GFP induction in TZM-GFP is observed on co-culture with experimentally infected macrophages. GFP-positive syncytium formation even by rare infected macrophages facilitates direct observation of infection in primary cells by electron microscopy. We show by limiting dilution that TZM-GFP cells can report a single infected macrophage, allowing quantification of cells that harbor infectious provirus. Co-cultured TZM-GFP with bronchoalveolar lavage (BAL) cells from asymptomatic HIV-positive Malawian adults reveals the induction of GFP and transfer of HIV from BAL cells of ART-treated and ART–naïve patients. Similarly using FISH:FACS, we find HIV mRNA predominantly in AMs at HIV diagnosis and in aviremic individuals on ART, suggesting the persistence of HIV in AMs despite successful therapy. Conclusions: Using FISH:FACS to build RNAseq profiles of purified HIV-infected cells and novel viral outgrowth assays, we are generating a complete toolkit to identify bona fide ART-durable reservoirs in the deep tissues of human patients. These novel tools can facilitate the direct demonstration of HIV infection even from rare cells within human biopsy samples. We conclude that (1) ART, though successful in clearing viremia, spares HIV- infected AMs. (2) BAL cells from ART-treated patients generate infectious virus, showing the lung is a site of HIV maintenance. (3) HIV in the lung is mainly found in AMs, a potential anatomical reservoir that remains understudied. Our future work will apply synthetic reporter RNA to enable live sorting of HIV-infected cells from bulk human tissue samples, enriching these cells for more accessible assays of viral outgrowth. This study reports on the lung as a site of viral persistence and productive infection in the face of effective ART, and will bring significant changes to the way we approach clearance of HIV tissue reservoirs with novel therapies and vaccines.

8 Beta-Cell GLP-1R Contributes to Improved Glucose Tolerance After Vertical Sleeve Gastrectomy in Mice Garibay D, McGavigan AK, Lee SA, Showalter AD, Michael MD, Sloop KW, Cummings BP Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain undefined. Post-operative increases in postprandial glucagon-like peptide-1 (GLP-1) secretion may contribute; however, previous work has been equivocal. To test the contributions of β-cell GLP-1 receptor (GLP-1R) signaling we used a β-cell specific tamoxifen-inducible GLP-1R knockout mouse. At 8wks of age, Glp-1rβ-cell+/+ (WT) and Glp-1rβ-cell-/- (KO) male mice were placed on a high fat diet (HFD) for 6wks and then switched to HFD supplemented with 400mg tamoxifen/kg diet for the rest of the study. Mice underwent sham or VSG surgery at 16wks of age and were then fed ad libitum (n=7-9). Mice underwent oral glucose tolerance testing (OGTT) at 3wks and were euthanized at 6wks after surgery. Body weight and food intake was reduced after VSG compared to the respective sham-operated control (p<0.05). Body weight and food intake did not differ between genotype for either treatment. OGTT data revealed an improvement in glucose tolerance in VSG WT, but not VSG KO. Furthermore, the glucose AUC was significantly elevated in VSG KO versus VSG WT (Glucose AUC0-120:Sham WT=26788±1296, Sham KO = 30273 ± 4521, VSG WT=22312±2439, VSG KO=30766±2799 mg·min/dl; p<0.05). The augmentation in glucose-stimulated insulin secretion during the OGTT was blunted in VSG KO versus VSG WT (OGTT percent increase in insulin from baseline to peak values: Sham WT=148±48, Sham KO=96±43, VSG WT=294±79, VSG KO=138±31%; p<0.05). Overall, our data suggests β-cell GLP-1R signaling contributes to improved glucose regulation and insulin secretion after VSG. 9 Anti-microbial properties of equine mesenchymal stromal cells Rebecca M. Harman and Gerlinde R. Van de Walle Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca NY Antibiotics (Abx) are commonly used in veterinary medicine to treat infectious diseases caused by bacteria and other microorganisms. The extensive use of Abx in recent years has led to the emergence and spread of Abx-resistant microorganisms. When resistance occurs, previously successful drugs are no longer effective, creating a need for alternative approaches to fight bacterial infections and/or to boost the host’s antimicrobial defense system. Mesenchymal stromal cells (MSC) are adult multipotent progenitor cells, that can be isolated, expanded in culture, and used therapeutically. MSC actively contribute to healing processes by promoting tissue regeneration, modulating immune responses, regulating inflammation and preventing pathological scar formation. Our lab has carried out in vitro experiments designed to study the therapeutic effects of equine MSC on healthy and dysregulated cell types present in lower limb wounds of horses presenting with exuberant granulation tissue (EGT). Our results suggest that MSC can act on resident skin cells to alter the wound environment, improve healing and reduce EGT. Since bacterial infection is known to promote the formation of EGT, we have started to examine the anti-microbial properties of equine MSC as well. Our findings show that MSC secreted products can prevent bacterial growth and the formation of biofilms in vitro, and MSC can kill internalized bacteria. MSC express genes coding for anti-microbial peptides (AMP), and expression of these genes can be upregulated in the presence of bacteria. Several AMP are detected in conditioned medium from MSC cultures. In addition to these direct anti-microbial effects, MSC protect skin cells from bacterial toxin- induced cell death, and stimulate resident skin cells to increase AMP production. Based on these results, we believe MSC will be a useful therapy for horse EGT, in part due to their direct and indirect anti-microbial properties.

12 HAP2-mediated cellular fusion in a sexual ciliate: what to look for in a gamete fusogen and what this means for eukaryotic sex Jennifer F. Pinello , Alex Liqi Lai, Donna Cassidy-Hanley, and Theodore G. Clark Department of Immunology and Infectious Disease, Cornell University, Ithaca, NY While the mechanisms underlying gamete cell fusion during sexual reproduction remain obscure, recent studies have implicated the conserved transmembrane protein, HAP2/GCS1, as an ancestral gamete fusogen. Despite the absence of sequence similarities with other known fusogens, genetic disruption of the HAP2 locus in a variety of species ranging from protists to flowering plants leads to a decisive block to fertilization due to an inability of gametes to catalyze membrane fusion events. To better understand the role of HAP2 in cell-cell fusion we developed a flow cytometry-based assay to assess membrane pore formation during conjugation in the model ciliate, Tetrahymena thermophila. This assay provided a streamlined method to investigate the functional significance of HAP2 domains. In the course of this work, we found the ectodomain of Tetrahymena HAP2 shares a high degree of predicted structural homology with the dengue virus E glycoprotein (a class II viral fusogen). This region contains a predicted peptide loop analogous to the viral fusion loop used to perturb target membranes at the initiation of virus entry into host cells. We found that deletion of the fusion loop or the entire dengue virus E glycoprotein homology domain from T. thermophila HAP2 completely eliminated fusion between mating Tetrahymena cells. In contrast, the HAP2 endodomain appeared largely dispensable for membrane fusion, as did ZFR1, a protein that localizes to the mating junction and is required for fertilization much like HAP2. Finally, biophysical studies with the predicted fusion loop suggested direct interactions of this peptide domain with artificial lipid bilayers. These findings raise interesting questions regarding the evolutionary origin of HAP2, and may lend important insights into the mechanistic function of this key developmental protein. 13 Two viral nonstructural proteins are sufficient to compartmentalize the host cell translational machinery in a virus-derived matrix Dania Villarnovo and Kristy Richards Field of Comparative Biomedical Sciences, Cornell University, Ithaca, NY Reoviruses compartmentalize translation within virus-induced inclusions called viral factories. Viral factories are the sites of viral replication, transcription, and assembly. Here we report that ectopic expression of two viral nonstructural proteins are sufficient to compartmentalize translation within viral factory-like structures that form within transfected cells.

14 The independent emergences of H3N8 and H3N2 canine influenza viruses and their evolutionary dynamics in the USA. Ian Voorhees , Edward Dubovi, Amy Glaser, Edward Holmes, and Colin Parrish Department of Immunology and Infectious Disease, Cornell University, Ithaca, NY Influenza A viruses (IAVs) are notorious for the ability to “spillover” from their reservoir hosts, waterfowl and shorebirds, to cause disease in other animals. While these cross-species transmission events are fairly common, they rarely result in sustained epidemics. Until recently, dogs were considered refractory to IAV infection. However, within the last 20 years, IAVs have successfully jumped into dogs twice independently, producing sustained canine-to-canine transmissions in both instances. H3N8 canine influenza virus (CIV) emerged in Florida from an equine IAV in 1999, while H3N2 CIV emerged directly from an avian IAV in China in 2006 and spread to domestic dogs in the USA in 2015. The emergences of the CIVs provide an opportunity to compare the evolutionary events that have enabled two recently emerged, distinct IAVs of different origins to convergently adapt to the same mammalian host. To this end, whole-genome amplification, sequencing, and phylogenetic analysis of the CIVs were conducted, enabling high-resolution reconstructions of the viruses’ evolutionary histories. Results indicate that, in both subtypes, viruses are maintained in areas of dense dog populations and high contact frequencies after their initial emergence. Additionally, extensive host contact heterogeneity and rapid evolutionary rate typical of IAVs have produced geographically distinct viral clades with potentially unique characteristics. The CIVs provide an excellent model in which to answer basic questions in viral evolution. Furthermore, given their global abundance and synanthropicity, canine hosts are ideally suited to act as evolutionary and ecological stepping-stones for IAV transfer to humans – a threat that should not be overlooked. 15 Defining Mechanisms of Lipid Utilization in Mycobacterium tuberculosis Using a Fluorescent Reporter of Cholesterol and Fatty Acid Metabolism Kaley Wilburn , Christine Montague, Evgeniya Nazarova, Thuy La, and Brian VanderVen Department of Microbiology and Immunology, Cornell University, Ithaca, NY B cells generate a repertoire of antigen-specific antibodies in response to immunization. Antibody-based immunotherapies hold great promise for treating cancer and other diseases in humans. However, for cats we need to identify species-specific B cell clones that produce antibody specific to the antigen of interest. From such clones, the mRNAs encoding the cognate heavy and light immunoglobulin (Ig) can be isolated, cloned and sequenced. Finally, with this information vectors that can express these antibodies in exogenous systems can be developed and the antibodies can be tested for therapeutic efficacy. Such approaches have been developed in human medicine. However, we lack information about feline Ig sequences and the feline IgG response to antigen. The goal of this study was to fully sequence Ig heavy and light chains from feline B cells and then to adapt human Ig expression vectors to allow ectopic expression of feline Ig molecules. Such vectors can then be used to insert variable heavy and light chain sequences that can confer different antigen specificities. We recovered mRNA from feline B cells from two cats and cloned and sequenced the variable and constant regions of the feline IgG1a, Igλ and Igκ sequences. The full-length feline IgG1a, Igλ and Igκ sequences were assembled and representative sequences were cloned into a bi-cistronic mammalian expression vector designed by Dodev et al (Scientific Reports, 4:5885, 2014). Here we report novel feline Ig sequences and on a technique to grow and clone feline Ig-secreting cells to allow identification of antigen-specific feline mAbs.