Common Fragile Site Flexibility Peak Does Not Strongly Induce Mitotic Recombination in S. cerevisiae

Valerie R. Sponyoe and Dr. Anne M. Casper, Eastern Michigan University, Ypsilanti, Michigan

Common fragile sites (CFS) are specific loci on chromosomes that display instability when exposed to conditions of replication stress. CFS are associated with the deletion of tumor suppressor genes and the amplification of oncogenes. It has been shown that breaks at a yeast fragile site stimulate repair through homologous recombination events that lead to loss of heterozygosity. Here, we have examined whether a sequence motif known as a flexibility peak found in human CFS FRA3B stimulates homologous recombination events when inserted onto a chromosome of the yeast Saccharomyces cerevisiae. Overall, it does not appear that the inserted FRA3B flexibility peak induces homologous recombination in yeast.

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A Descriptive Examination of Signal Property Vectors from Open Reading Frames Assigned Acetohydroxy Acid Synthase Function in Methanocaldococcus jannaschii

Michael Hourani and Laura K. Harris, Department of Science, Davenport University, Lansing, Michigan

Several studies have examined the relationship among nucleotide, absolute codon, and amino acid frequencies from open reading frame (ORF) numbers 0277 and 0063, thought to encode large sub-units of acetohydroxy acid synthase (EC 4.1.3.18, AHS) in Methanocaldococcus jannaschii. These studies have identified a difference between ORF MJ0277 whereas MJ0663 did not show a difference with the rest of the M. jannaschii genome with one study comparing these signal properties to those 27 other organismal genomes, concluding that ORF MJ0277 may have derived from an external origin. Ergo, this study aimed to verify and expand upon previous conclusions by analyzing the nucleotide, absolute codon, and amino acid frequency vectors using NCBI sequences. First was an examination of MJ0277 and MJ0663 with M. jannaschii genome. We show an almost 1-fold increase in c2-squared values for all MJ0277 frequency vectors over MJ0663 when compared to the M. jannaschii genome. The difference between MJ0663 and the M. jannaschii genome for all frequency vectors were similar in difference to the ilv genes in E. coli compared to the E. coli genome. This confirms the findings of prior studies. Furthermore, to examine which archaeal organism MJ0277 may have originated from frequency vectors were compared to those of 16 fully sequenced genomes. For MJ0663, M. jannaschii appeared the best fit with the smallest c2-squared values for most signal properties. For MJ0277, several organisms had lower c2-squared values for nucleotide and amino acid frequency vectors, though only one organism, Staphylothermus marinus F1 had a lower absolute codon frequency value. The shift in absolute codon frequency toward that of the M. jannaschii genome without a similar shift in nucleotide and amino acid frequencies as seen by the number of archaea with lower c2-squared values, support the conclusion that MJ0277 is derived from an external origin and adapting to the M. jannaschii tRNA set.

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Random Mutagenesis of Autophagy Related Protein 9 (Atg9)

Bridget Kennedy, Eastern Michigan University, Ypsilanti, MI

Autophagy is a mechanism used to rid eukaryotic cells of malfunctioning proteins and organelles by sending them to the lysosomes to be degraded. Autophagy thus acts as a cellular "garbage disposal" that helps prevent neurodegenerative diseases like Parkinson's and Alzheimer's. Despite its importance for human health, no one really understands how autophagy works. One key autophagy protein conserved across species is Atg9. We are preparing to do random mutagenesis followed by a yeast screen to determine which amino acids of Atg9 are the most important. We are beginning with site-directed mutagenesis to target a specific, particularly interesting region of Atg9 for the screen.

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Investigating Gene Conversion Stimulation by Fragile Site FS2

Mikael Dunn and Anne Casper, Eastern Michigan University, Ypsilanti, MI 48197

Partial inhibition of DNA polymerase causes breaks at specific sites called fragile sites. We hypothesize that instability at fragile site FS2 in Saccharomyces cerevisiae in cells under replication stress is repaired by homologous recombination (HR), resulting in gene conversion (GC). To test this, we identified cells that repaired a break by HR during mitosis. For this identification, we used a system thatwhen the cell has repaired a break by HR, it produces a colony with a white half and a red half. We screened 34,975 colonies and found 201 sectored colonies, of which 43 are GCs, accounting for 22% of all HR events. This supports our hypothesis that GC events are stimulated by FS2 instability under replication stress.

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Immunohistochemistry of Breast Cancer for TH1 verses TH2 Response

Tayson T. Lin, Dana L. Schalk, Archana Thakur, and Lawrence G. Lum
Karmanos Cancer Center, Detroit, MI 48201

The immune system plays a critical role in cancer detection and eradication. However, the body’s immune defenses are imperfect because tumors still develop. There are multiple mechanisms by which cancer can escape immune detection and subsequent attack. For instance, the tumor microenvironment could suppress anti-tumor Cytotoxic T- Cells through recruiting immunosuppressive Type 2 Helper T Cells (TH2). Conversely, activity of Type 1 Helper T Cell (TH1) is attributed to Cytotoxic T-Cell activation and tumor killing. Previous studies documented that infiltrating white blood cells around primary breast tumor sites as well as the presence of immunostimulatory TH1 responses was attributed with reduced cancer recurrence and longer survival. However, these studies did not simultaneously examine the patient’s auxiliary lymph nodes, which are removed as a part of the surgical management of invasive breast cancer. In this project, we developed an immunohistochemistry assay that images interferon-gamma (IFN-γ) and interleukin-10 (IL-10), which are representative of TH1 versus TH2 activation, respectively, in paired breast cancer and sentiel lymph node biopsies. Currently, we have successfully imaged biopsies from 24 patients and are in the process of conducting immune evaluations. We hypothesize that this assay will lead to improved understanding of the phenotype and function of TH1 versus TH2 T cell subpopulations in both breast tumors as well as the sentiel lymph nodes.

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Microbial Community Analysis in Petroleum-Contaminated Sediments Displaying High Magnetic Susceptibility

Anja Williams, Carol Beaver, Estella Atekwana2, Lee Slater3 and Silvia Rossbach, Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, 2Oklahoma State University, Stillwater, OK 74078, 3Rutgers University-Newark, Newark, NJ 07102

Terrestrial oil spills are a common heath threat to environmental and public health. Physical cleanup can increase toxic hydrocarbon exposure to the surrounding environment and local human populations, and does not completely degrade the hydrocarbons. Natural microbial biodegradation is an increasingly incorporated method of hydrocarbon mineralization that can convert aromatic and alkane hydrocarbons to CO2, H2, and CH4. However, this technique needs to be monitored to ensure effective cleanup. Magnetic susceptibility (MS) is a geophysical method that may observe changes in subsurface sediment magnetism caused by microbial activity, specifically anaerobic iron reduction that induces magnetite formation. Collaborators have seen an increase in MS within an aged, subsurface oil plume compared to sediments outside the plume. Furthermore, the high input of carbon from an oil spill makes other required nutrients, like nitrogen, limiting. Nitrogen fixing microorganisms may be providing useable forms of nitrogen for hydrocarbon degraders. The first goal of this study was to characterize the microbial communities present at high and low levels of MS with 16S rRNA gene clone libraries, and identify the organisms degrading oil and inducing the high magnetism. Clones similar to Albidiferax and Desulfosporosinus were found indicating iron reduction is occurring, which could be inducing the increase in magnetite.  Clones similar to Smithella and Methanogregula were also found indicating methanogenic hydrocarbon breakdown. The second goal was to characterize the nitrogen fixing community present at the oil plume with nifH gene clone libraries,and identify the organisms capable of producing a useable form of nitrogen. Clones similar to rhizobia were found as well as other microorganisms associated with hydrocarbon breakdown. These results suggest that an increase in MS is directive towards active hydrocarbon breakdown using iron reduction, and that nitrogen fixation may be occurring by microorganisms not involved in hydrocarbon degradation as well as those that are.

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The expression of mouse Interleukin-2 by Tanapoxvirus attenuates virus replication

Tiantian Zhang, Dennis H. Kordish, and Karim Essani, Western Michigan University, Kalamazoo, MI, USA, 49008

Interleukin 2 (IL-2) is a pleiotropic cytokine that plays a key role both in innate and adaptive immune systems. It has been shown to be involved in the differentiation of T cells and activation of NK cells. It has been observed that mammalian viruses expressing IL-2 are attenuated. To evaluate the role of IL-2 in virus replication, we have generated a tanapoxvirus (TPV) mutant (TPVΔ66R/mIL-2) where the 66R/thymidine kinase (TK) gene has been replaced with mouse interleukin-2 transgene. In cell culture, the replication of TPVΔ66R/mIL-2 was more than 10 fold lower than that of both wild-type TPV (wtTPV) and TPVΔ66R at a low multiplicity of infection (MOI). The replication of TPV-GFP was about 4 fold lower when cells were co-infected with TPVΔ66R/mIL-2, as compared to co-infection experiments using TPVΔ66R. Addition of monoclonal antibody anti-mIL-2 to the medium failed to enhance the replication of TPVΔ66R/mIL-2 in cell culture, and addition of recombinant mIL-2 to the medium did not inhibit the replication of wtTPV or TPVΔ66R virus in cell culture. Taken together, these results suggest that IL-2 reduces the virus yield by an unknown mechanism when IL-2 is expressed intracellularly in virus-infected cells.

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Development of Dominant Negative NOD1 and NOD2-expressing Transposons for Tissue-specific Manipulation of Zebrafish (Danio rerio) Innate Immune System using Gal4/UAS-mediated Expression

Kevin Stille, Kacie Gee, Jamaal Tarpeh, Kendra Garcia, Emerald Butko2, Natasha DelCid2 and L. Rob Peters, Aquinas College, Grand Rapids, MI, 49506, 2University of California, San Diego, La Jolla, CA, 92093

NOD1 and NOD2 of Danio rerio are orthologs to the human genes encoding cytosolic innate immune pattern recognition receptors (PRRs) that bind bacterial cell wall components. In humans, mutations in these genes are highly correlated with inflammatory intestinal diseases such as Crohn’s disease. There are still many unanswered questions regarding the processes leading to these diseases. Danio rerio embryos are a unique model system with natural transparency and ex vivo development that are easily adapted to high-throughput analyses. We plan to create tissue-specific expression vectors containing dominant negative NOD receptors based on those characterized in mammalian studies. These tools will allow the study of the receptors in the development of the innate immune system and their interactions with the intestinal microbiota. Additionally, further characterization of the role of NOD1 and NOD2 in interactions between the innate immune system and the intestinal microbiota may lead to a better understanding of the etiology of inflammatory intestinal diseases like Crohn's disease.

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Cloning of cpnC and cpnD cDNAs in Dictyostelium

Bria N. Graham, Dexter R. McKellar, and Cynthia K. Damer, Central Michigan University, Mount Pleasant, MI 48859

Environmental Signaling and Biofilm Formation in Escherichia coli

Adam Pickrum, Jordan Zhou, Steve Wilkinson, and Aaron Baxter, Grand Valley State University, Allendale, MI, 49401

The ability of bacteria to form a biofilm can be considered one of the most important virulence factors they have.  A biofilm acts as a platform for bacterial communication during pathogenesis and makes the bacteria more resilient to therapeutic agents, such as antibiotics.  Biofilm formation is under strict regulation that involves many genes.  Gene expression is dependent on various environmental signals including temperature, pH, and oxygen levels. We hypothesize that the ability of Escherichia coli to form a biofilm differs between aerobic and anaerobic environments.  Our efforts have resulted in the development of a biofilm assay capable of determining the percent biofilm formation.  Also, our group is creating a growing library of E. coli mutants.  Random mutations are made in the genome of E. coli strain MG1655 by transposon mutagenesis.  The goal of this project is to identify genes that contribute to biofilm formation differently in aerobic and anaerobic conditions.

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Biodiesel Glycerol Byproduct as Feedstock for Pseudomonads of Industrial Significance in Undergraduate Research

James F. Graves, Christopher J. Fronczak, Kezia S. Mathew, and Seon Gyeong Park, University of Detroit Mercy, Detroit, MI 48221

In manufacture of biodiesel a byproduct is made that contains glycerol.  Pseudomonas species are used to synthesize biochemicals and in bioremediation.  Because some isolates of Pseudomonas utilize glycerol the growth of selected species with biodiesel glycerol byproduct was investigated.  When assessed by a rapid solid phase lysochrome diazo dye assay (Precision Labs, Inc.), glycerol, byproduct made by transesterification or catalyst column, and soap failed to test positive for lipid in contrast to vegetable oil, biodiesel and tween 20.  On spirit blue agar Pseudomonas species exhibited a halo around areas of bacterial growth which was indicative of the presence of lipase.  Ability to grow at 14oC or 42oC differentiated several species.  Antibiograms made by disk diffusion showed all the bacteria exhibited sensitivity to ciprofloxacin, gentamicin and tetracycline, but some grew up to disks of chloramphenicol and erythromycin.  On minimal agar, containing only essential nutrients, with glucose, glycerol or byproduct made by transesterification or catalyst column as carbon sources, P. aeruginosa, P. cepacia, P. fluorescens and P. putida showed fair to excellent growth.  In minimal broth, when glucose, glycerol or byproduct from catalyst column served as carbon source, measurement of optical density (OD) by a Klett – Summerson colorimeter showed P. aeruginosa, P. fluorescens and P. putida attained ODs over 150. With byproduct made by transesterification, P. aeruginosa, and P. putida produced ODs near 100 and P. fluorescens showed an OD near 50.  Viable cell counts in medium with byproduct attained 108 colony forming units (CFU)/ml.  When tested in a microplate redox-based array (Biolog, Inc.) the bacteria demonstrated ability for oxidation of glycerol and tweens.  Biodiesel glycerol byproduct could serve as a possible carbon source for Pseudomonas species.

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Lactobacillus rhamnosus exhibits native adhesin-mediated coaggregation with Bacteroides species

Samuel J. Schotten and Daniel Clemans, Eastern Michigan University, Ypsilanti, MI 48197, Eric Martens and Nathan Porter, University of Michigan Medical School, Ann Arbor, MI 48109

The GI tract of mammals is host to a rich diversity of bacteria that interact by coaggregation to form biofilms.  Bacteroides fragilis and Bacteroides thetaiotaomicron are two of the most abundant occupants of the human large intestine.  These species may interact with the probiotic strain, Lactobacillus rhamnosus GG, a strain that improves host immunity and nutrient metabolism and absorption.  This study sought to characterize the coaggregation interaction of L. rhamnosus GG with wild and capsular type B. thetaiotaomicron VPI 5482, as well as B. fragilis ATCC 15285.  Cultures were grown in a variety of liquid media, extracted by centrifugation, and suspended in a standard coaggregation buffer.  Mutants strains of B. thetaiotaomicron only expressing a single exopolysaccharide capsular type or no capsule were provided to the lab for more detailed assessment of coaggregation.  Assays were performed on the mixed cell suspensions to assess observations of cell aggregation by a 4-point scale under standard conditions, and further, after treatment of the suspension with heat and protease.  Results showed moderate scores for L. rhamnosus from Todd Hewitt media with B. fragilis from RCM media,and also with B. thetaiotaomicron from TYG and various isolates of its mutant capsular types.  Although heat assays displayed an unexpected strengthening of coaggregation after treatment, protease assays displayed scores of no coaggregation after treatment of L. rhamnosus.  It was concluded that L. rhamnosus likely contains the adhesin involved in coaggregation on its cell surface, and it binds to a ligand on the surface of B. thetaiotaomicron.

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CpnA Binds to Actin Filaments in a Calcium-dependent Manner In Vitro

Matthew J. Buccilli, Bryana L. Borders, Andrew A. Banas, Mingxi Han, Hanqian Mao, David Loiselle*, Timothy A.J. Haystead*, and Cynthia K. Damer, Central Michigan University, Mount Pleasant, MI 48859, *Duke University Medical Center, Durham, NC 27708

Copines make up a multigene family of calcium-dependent, phospholipid-binding proteins. Copine proteins consist of two C2 domains at the N-terminus followed by an “A domain” similar to the von Willebrand A domain found in integrins. The C2 domain is a calcium-dependent membrane-binding motif, while the A domain is thought to be a protein-binding domain. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA- cpnF. Previous research showed that cpnA− cells exhibit a cytokinesis defect, a developmental defect, and a defect in contractile vacuole function. To fully understand the role of CpnA in these cellular processes, we used column chromatography and mass spectrometry to identify proteins that interact with CpnA.  One of the proteins identified was actin. To determine if CpnA associates with the actin cytoskeleton, we treated cells expressing GFP-CpnA or GFP-Ado (containing the A domain of CpnA) with Triton X-100 and spun down the insoluble cytoskeletal fraction. GFP-CpnA was found in the cytoskeletal pellet only in the presence of calcium, while GFP-Ado was found in the cytoskeletal pellet in the presence and absence of calcium. To determine if CpnA directly binds to actin, we performed F-actin binding assays with purified GST-CpnA and found that GST-CpnA bound to actin filaments in a calcium-dependent manner. In addition, we performed immunoprecipitations with cells expressing GFP, GFP-CpnA, and GFP-Ado. We then incubated the precipitated proteins with F-actin or G-actin in the presence or absence of calcium. We found that F-actin, but not G-actin, co-precipitated with GFP-CpnA in the presence of calcium. Our results indicate that CpnA binds directly to F-actin only in the presence of calcium.

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Analysis of Tau Expression in a Novel Tauopathy Model System

Zachary W Johnson, Erwin, Amanda L, Miller Kristi E, Steinhilb, Michelle L, and Damer Cynthia K., Central Michigan University, Mt. Pleasant, MI 48859

Alzheimer’s disease (AD) is the sixth leading cause of death in the United States. This neurodegenerative disorder impairs brain regions associated with learning and memory. AD is characterized by two brain features: amyloid plaques and neurofibrillary tangles. Neurofibrillary tangles are intracellular aggregates of the protein tau, a microtubule binding protein. Abnormal phosphorylation and proteolysis of tau in AD brains is thought to be toxic. We are using the eukaryotic singled-celled model organism Dictyostelium discoideum to study tau toxicity. The goal of this study is to investigate the progression of tau toxicity using four different tau constructs: full-length human wild-type tau (tauWT), incompetent phosphorylation tau (tauAP), calpain resistant tau (tauCR), and a proteolytic17kDa fragment of tau (tau17kD).  Previous results showed that constitutive expression of tauAP in Dictyostelium cells was not toxic, while expression of tauWT, tauCR, and tau17kD resulted in cell death within 2-3 weeks. Therefore, we are now using a doxycycline-inducible expression system in Dictyostelium.  We have induced expression of tauAP and tau17kD in Dictyostelium thus far. Our results indicated that both tauAP and tau17 can be expressed in a doxycycline dose-dependent manner. In addition, tauAP is transiently expressed with one dose of doxycycline, peaking at 24-48 hours after induction, and is then rapidly degraded. In contrast, tau17kD is still detected in cells seven days after one dose of doxycycline. These data suggest that Dictyostelium cells are able to rapidly degrade tauAP, most likely through proteasomes, but not tau17kD. When tau17kD expression is induced with doxycycline continuously, cells slowly began to die over a 2-3 week period. Ultimately, we plan to use this new tauopathy model to reveal which posttranslational modifications cause tau toxicity and explore the mechanism of toxicity.

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Tanapoxvirus Ankyrin Repeat Genes and Tumor Selectivity

Yogesh Suryawanshi and Karim Essani, Western Michigan University, Kalamazoo, MI 49008

Standard cancer treatments have succeeded in improving post-treatment life expectancy among cancer patients but failed to effectively cure the disease. Moreover, most standard cancer treatments that are currently available lack target specificity against cancer cells and usually lead to severe adverse effects that worsen the quality of life in cancer patients. Oncolytic virotherapy is an emerging treatment option that utilizes viruses that can selectively replicate in cancer cells, leading to their destruction. Ankyrin (ANK) repeat genes have been well conserved among orthopoxviruses and several poxvirus ANK proteins share general structure, including the F-box like motif at the carboxyl terminus. The F-box motif in poxvirus ANK proteins have been shown to interact with cellular ubiquitin ligase complexes which can result in ubiquitination of associated host proteins. Poxvirus ANK-F-box motif appears to play critical role during viral replication by interacting with host cell proteins through ubiquitin-mediated degeneration. Poxvirus ANK proteins are also known to inhibit apoptosis of infected host cells and inactivate host immune response. Cancer cells also establish anti-apoptotic and anti-cancer immune response mechanisms. Our hypothesis is that ablation of ANK repeat genes from TPV can possibly render the virus incompetent to replicate in normal cells. In this study, we have targeted the 146R, an ANK repeat gene in TPV which is characterized by an F-box motif at the carboxyl terminus. TPV mutant (TPVΔ146R) generated by selective deletion of 146R gene was tested for selective replication in human cancer cells, using a panel of human colorectal and ovarian cancer cells. . TPVΔ146R demonstrated some degree of selectivity in human ovarian cancer cell line (SW626) and human colorectal cancer cells (HCT 116 and Caco-2), as compared to the wild type TPV (TPV-Kenya). The precise mechanisms involved in providing selectivity to the TPVΔ146R in some of these cancer cell lines remain to be elucidated.

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Exploring the Functional Relationship Between Copine A and Actin Filaments in Dictyostelium

Samantha P. Perry, April N. Ilacqua, Thomas S. White, Tolga Kaya, and Cynthia K. Damer, Central Michigan University, Mount Pleasant, MI 48859

Copines are family of proteins found in many eukaryotic organisms, including single-celled Dictyostelium. Copines bind to membranes in a calcium-dependent manner, but their exact function is unknown. In previous studies, Dictyostelium cells lacking the cpnA gene exhibited several developmental defects. Cells were delayed in aggregation and formed larger than normal slugs that demonstrated altered phototaxis and thermotaxis and did not develop into fruiting bodies. Previous studies also indicated that CpnA is able to bind actin filaments. In this project, we are using cpnA- cells to test the hypothesis that CpnA functions in the regulation of the actin cytoskeleton. To determine if there is a difference in the amount of actin filaments in wild-type cells (WT) and cpnA- cells, we treated cells with Triton X-100 and pelleted the insoluble actin cytoskeleton. The pellet and supernatants were analyzed using Western blots with an antibody to actin.  We then used densitometry to quantify the actin bands. We found no difference between the amount of actin filaments in cpnA- cells and WT cells. Next we used rhodamine-phalloidin to stain actin filaments and DAPI to stain nuclei in both WT and cpnA- cells. We then counted the number of nuclei and actin filament clusters in individual cells. We found that there was no difference in the number of nuclei between cell types, but there were twice as many actin filament clusters in cpnA- cells than WT. Next we performed chemotaxis assays. We found that the cpnA- cells moved towards folate more slowly than WT cells. In contrast, cpnA- cells appeared to sense the cAMP and move, but did not move towards cAMP, while WT cells moved towards cAMP. Our results indicate that CpnA plays a role in regulating actin filaments and this function may be important to CpnA’s role in cAMP chemotaxis.

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Investigation of Copine Protein Localization in Dictyostelium discoideum

April Ilacqua, Price JE, and Damer CK, Central Michigan University, Department of Biology, Mount Pleasant, MI 48859

Coaggregation between Lactobacillus rhamnosus and other Gut Microbes

Jamal A. Alhabeil, Samuel J. Schotten, Dr. Eric Martens,b Nicole Koropatkin,b Nicholas Pudlo,b Dr. Daniel Clemans, Eastern Michigan University, Ypsilanti, Michigan, 48197, University of Michigan, Ann Arbor, Michigan, 48109b

Within the Human Gastrointestinal Tract (GIT), various microorganisms coaggregate with each other and often form biofilms, a grouping of microbes protected by an outer layer of slime. The ability to form and utilize biofilms is integral for the survival of many of these gut microbes. This study set out to test and identify which factors played a role in the coaggregation of various representative gut microbes. Lactobacillus rhamnosus GG (ATCC 53103), a known biofilm producer, was grown on a modified Tryptic Soy medium to encourage coaggregation. It was mixed together with other gut microbes under various conditions (glucose, protease, and heat inhibition) in hopes of reducing its coaggregation ability. Glucose and heat inhibition assays had no major effect on L. rhamnosus coaggregation, while the protease inhibition assay resulted in a decreased ability to coaggregate with the other microbes. Membrane-bound proteins are most likely the key elements present in L. rhamnosus coaggregation.

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Analyzing the Roles of Rfg1 and Tup1 in the Interactions Between Candida albicans and Other Microbes

Dorothy D. Butler, Engerson, Andrea A. and Thomas, Derek P., Grand Valley State University, Allendale, M.I. 49401
                                       
Nosocomial Candida albicans infections are on the rise in the United States, primarily due to an increased number of invasive procedures, transplants, and use of broad range antibiotics and immunosuppressive agents. One important virulence factor in Candida species is its ability to transition between two morphologies, yeast and filamentous cells. Filamentous formation is controlled by several transcription factors that induce filamentation and several negative regulators that repress filamentation. Rfg1 is one of several partner proteins thought to function in combination with Tup1 to repress genes associated with filamentation and thus potentially influence the virulence of Candida.  Here we investigate the negative regulators Tup1 and Rfg1 in Candida and their effect on interactions between Candida albicans and various bacteria. In their natural environment, bacteria and unicellular eukaryotes are found together exhibiting both synergistic and antagonistic interactions. Previous studies in our group have documented decreases in the levels of both RFG1 and TUP1 when wild-type Candida was grown near Acinetobacter baumannii. Interestingly, during such bacterial-fungal coexistence the levels of Rfg1 and Tup1 are not consistent with the action of a repressor of filamentation. These results suggest TUP1 and RFG1 are impacted by cellular signals that form part of the interactions between Candida and other commensals. Here, we looked at over-riding such decreases and how this influences the effects of bacteria on the morphology and hyphal specific gene transcription of Candida when grown as a biofilm.

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Analyzing the role of a putative phosphatase 2A component in C. albicans

Julie Cole, Elizabeth A. Sommers, Ian A. Cleary‡, and Derek P. Thomas, Grand Valley State University, Allendale, MI 49401, ‡ University of Tennessee at Martin, Martin, TN 38238

Candidiasis now represents the fourth most frequent nosocomial infection both in the US and worldwide. Candida albicans is the most common cause of candidiasis, the pathogenic potential of which is intimately related to certain key processes, including filamentation. The transcriptional repressor Nrg1 has been shown to be closely linked to the control of both filamentation and pathogenicity. Proteomic studies have shown that the gene 19.1468 is altered during filamentation. 19.1468 is thought to encode a protein similar to Cdc55p which is one of two regulatory subunits of the Phosphatase 2A (PP2A) complex, and plays an important role in cell cycle progression and filamentation. S. cerevisiae strains that lack Cdc55p are defective for filamentous growth. Here we demonstrate that an over-expression of Cdc55p is sufficient to override Nrg1-mediated repression; this over-expression drives filamentation in a variety of conditions. We used Real-time PCR analysis to examine the regulation of filamentation-associated genes during Cdc55p driven filamentation in the presence of high levels of Nrg1p.  Furthermore, we demonstrate that when compared to a strain over expressing just NRG1 this strain restores virulence in the Galleria mellonella infection model. We are now further examining the specific contributions of this protein to hyphal development in C. albicans.

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Oncolytic Tanapoxvirus Lacking ORF 2L Reduces Human Ovarian Cancer Tumor Load in Nude Mice

Jonathan D. Hoerger, Yogesh Suryawanshi, Emily Byers, and Karim Essani
Western Michigan University Kalamazoo, MI 49008

Ovarian cancer (OC) is the leading cause of death among all gynecological cancers with an estimated 14,270 deaths in the U.S. in 2014.  The 5-year survival rate for OC is 44.6%, with the current standard treatment options.  A promising option of treatment termed oncolytic virotherapy (OV) is being developed for many different types of cancer including OC. Tanapoxvirus (TPV) is an antigenically distinct poxvirus, and is limited to equatorial Africa. Patients around the globe do not have prior immunity to TPV unlike most OVs currently under development.  TPV has been tested against human colorectal cancer (hCRC) and has demonstrated an ability to regress cancer growth in nude mice xenografted with hCRC. 
TPV was genetically modified to target carcinomas, which typically have increased levels of thymidine monophosphate (TMP) the product of thymidine kinase (TK), by deleting the viral TK gene (66R).  In this study, we have tested a number of TPV recombinants (TPV/egfp, TPV/∆2L, TPV/∆2L/∆66R, TPV/∆2L/∆66R/flic, TPV/∆66R/flic, TPV/∆66R) for their ability to replicate in the human ovarian cell line A2780.  These viruses were also tested for their ability to decrease cell viability in the cell line A2780.  The positive results of the in vitro work lead logically to test the same viruses against the same cell line (A2780) in induced xenografts in nude mice.  Once the tumors reached 75 mm3, mice were randomly assigned to groups for treatment (mock or recombinant TPV at 5 x 106 pfu/100 µL intratumorally).  The tumor volume was measured every 3 days.
The cell viability assay showed that TPV decreased cell viability by ~40% in an 8 day period.  The virus replication assay showed that in general most of the viruses were capable of replicating ~20 fold in 8 days.  The mouse xenograft model showed that TPV/∆66R, and to a much greater extent TPV/∆2L, were capable of significantly decreasing tumor growth rates, with TPV/∆2L/∆66R trending towards significance.  Our results suggest that TPV is an effective OV in vitro and TPV/∆2L shows tumor burden reduction in vivo.  Support for this study was provided by a FRACAA grant from WMU.

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Investigation into the Role of Peptidase Apr1p in the Opportunistic Pathogen Canidida albicans

Christopher T. Howard and Derek P. Thomas, Grand Valley State University, Allendale, MI 49401

Worldwide, the opportunistic pathogen Candida albicans is the predominant cause of candidiasis and imposes a heavy human health and economic burden upon the global community. In the human body, C. albicans is capable of growth in a wide range of anatomical locations partly due to its ability to survive within a broad spectrum of oxygen availabilities.
In Saccharomyces cerevisiae, PEP4 codes for the vacuolar endopeptidase proteinase A (PrA). PrA is responsible for the activation of other vacuolar hydrolases and provides S. cerevisiae with increased resistance to reactive oxygen species (ROS). In C. albicans, the protein with the most similar polypeptide sequence to PrA isApr1p. Despite this similarity, Apr1p deletion has no detectable effects on the levels of other common active vacuolar hydrolases in C. albicans. Furthermore, although no relationship between PEP4 and the hypoxic response of S. cerevisiae has been observed, a recent study suggests that Apr1p is associated with the hypoxic response of C. albicans. As many of the environments encountered by C. albicans in the human body are hypoxic, any protein associated hypoxic response has the potential to be vital for the survival of the opportunistic pathogen in vivo.
Here we examine whether Apr1p has a function in C. albicans different from that of PrA in S. cerevisiae. We also explore whether the function of Apr1p is related to oxygen availability. To accomplish this we exposed various strains of C. albicans, including Δapr1/Δapr1, to high levels of extracellular ROS and various levels of oxygen availability. In addition, we examine whether Apr1p demonstrates increased expression in C. albicans hyphal cells as compared to yeast cells and whether the protein is potentially a virulence factor in the Galleria mellonella host model.

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Toxicity Mechanisms of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonic Acid (PFOS) on Rhodobacter sphaeroides

Erin Mulroney and Britt Ranson Olson, Lake Superior State University

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are man-made environmental contaminants that have been identified in numerous biological sampling projects. There is a need to investigate the potential effects that these perfluorinated compounds (PFCs) have on a cellular and genetic level because of their extreme persistence. Rhodobacter sphaeroides is a good model to demonstrate such effects because it has been shown to be sensitive to both PFOA and PFOS and it possesses similar structure and metabolic processes to those found in eukaryotic mitochondria. After separate PFC treatments and mechanical lysis, a higher concentration of PFOA was found in the cell contents of R. sphaeroides than PFOS. This was also reflected in the relative supernatant measures; PFOA is more readily taken up by the cell than PFOS is. A bioinformatics analysis of R. sphaeroides gene-chip data demonstrated changes in gene expression induced by both PFOA and PFOS, the most up-regulated being those involved in membrane transport. In showing that these PFCs, especially PFOA, can be moved into the cell, these results broaden our understanding of the potential cellular effects of exposure, as well as further support differing mechanisms of toxicity for these PFCs.

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