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ABSTRACTS

Western PA Undergraduate Biology Symposium

2 April 2016 Seton Hill University

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1. The Prevalence of Toxoplasma gondii in indoor and outdoor cats of Southwestern Pennsylvania Rachel Narasimhan and Dr. Wayne Rossiter Waynesburg University, Waynesburg, Pennsylvania The protozoan parasite Toxoplasma gondii may possibly be the most common parasite found in developed countries. The health risks the parasite poses to humans are wide ranging, from lymphatitis to fetal abnormalities. Because domesticated cats act as the only definitive host, the prevalence of infection in cat populations has been extensively studied. We hypothesized that outdoor, and more specifically rural cats would demonstrate a higher prevalence of infection due to their increased contact with possible intermediate hosts (other warm-blooded animal, namely, mice in the genus Peromyscus) and their increased likelihood to ingest raw or undercooked food. In coordination with the Humane Society of Greene County, PA, fecal samples were collected from indoor and outdoor cats found in both rural and suburban environments in Southwest Pennsylvania. Fecal flotations were utilized to detect the occurrence of oocysts in the sample, indicating infection.

(Oral Presentation and Poster)

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2. Multidimensional NMR techniques used to elucidate the structure of bioactive hydroheptelidic acid isolated from a Quercus endophyte McCullough, Tyler M.1; Valenti, Domenic2; Harper, James K.2 Department of Chemistry, Washington & Jefferson College1 Department of Chemistry, University of Central Florida2 Natural products derived from microbes found in living plant tissues (i.e. endophytes), are a viable and relatively unstudied source of novel compounds with medicinal and agricultural applications. Particularly, natural product research is desirable for the production of antibiotics and agrochemicals which are highly effective, yet may produce less environmental impact and possess lower toxicity than similar compounds produced through synthetic methods. Despite the lower cost of drug production through synthetic methods, large drug companies often pursue natural product research as it can produce unusual model compounds for further modification. From an endophyte cohabitating in a species belonging to the genus Quercus, hydroheptelidic acid was isolated and purified through separation chemistry techniques. The structure of the purified compound was characterized through electrospray ionization mass spectrometry and 2-D NMR spectroscopy techniques including COSY, HSQC, HMBC, and INADEQUATE. Other research indicates that hydroheptelidic acid is bioactive with potential medicinal and agricultural applications. (Oral Presentation)

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3. The Role of STAT4 and IRF5 Single-Nucleotide Polymorphisms on Susceptibility to Systemic Sclerosis Zachary Sheffler, Jamie Fornsaglio Seton Hill University, Greensburg, PA Systemic sclerosis (Scleroderma), a connective tissue disorder (CTD), is an autoimmune disorder characterized by the buildup of scar tissue in the skin and organs, vasculopathy in small blood vessels, and immune dysregulation with production of autoantibodies. Currently, no cure exists for scleroderma, and its pathogenesis is not well-understood. Since morbidity and mortality rates remain high, investigating the pathogenesis of scleroderma is crucial for identifying new therapeutic approaches. Research suggests that the dysregulation of type-I-interferon (IFN) and IFN-inducible genes, like IRF5 and STAT4, play a role in the pathology of scleroderma. Moreover, independent studies show that single nucleotide polymorphisms (SNPs) in IRF5 (rs4728142) and STAT4 (rs7574865) may be predictive of one’s prognosis or predisposition to the disease, respectively. As both the STAT4 and IRF5 genes encode proteins that are key activators of IFN signaling pathways, we hypothesized that scleroderma-associated alleles in the rs7574865 and rs4728142 single-nucleotide polymorphisms would act with additive effects on susceptibility to scleroderma. Genotypes were determined through DNA isolation from saliva and using custom TaqMan assays with the ViiA 7 real-time PCR system. Interestingly, each scleroderma patient had at least one risk allele for rs4728142 and at least one for rs7574865. Additionally, the risk alleles were equally frequent in scleroderma patients for both SNPs, at 66.7%. Due to small sample size and statistical insignificance, we could not infer if the SNPs acted additively with regards to scleroderma susceptibility. While this study suggested rs4728142 and rs7574865 may be associated with scleroderma, more work needs to be done. (Oral Presentation)

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4. LRP1 Signaling in Hepatocytes Following Bile Duct Ligation Zachary Sheffler,Yuhua Xue, Wendy Mars University of Pittsburgh, Pittsburgh, PA The low density lipoprotein receptorrelated protein 1 (LRP1) is a versatile cell surface receptor expressed ubiquitously throughout the body. With over thirty distinct ligands, LRP1 is involved in a multitude of physiological processes, including: endocytosis, signal transduction and transdifferentiation. Previously we noted the presence of phosphoLRP and its ligand, tPA, in hepatocytes adjacent to proliferating bile ducts following bile duct ligation (BDL). Contemporary studies show that under compromised biliary regeneration, hepatocytes are able to transdifferentiate into biliary epithelial cells (BEC).Consequently, we hypothesized that LRP1 might play a role in hepatocellular transdifferentiation to BEC. Here, using hepatocellular conditional LRP1 knockout (cKO) mice, we present evidence that LRP1 is required for the proliferation and/or transdifferentiation of hepatocytes into BEC following BDL.Knockout and wildtype (WT) mice were subjected to the irreversible biliary injury model of BDL to encourage proliferation/transdifferentiation. Elevated bilirubin levels in the blood confirmed cholestasis. Immunohistochemical staining for Ki67 (proliferation marker) in paraffinembedded samples revealed bile ducts proliferated extensively in WT, but not KO, mice subjected to BDLs. Western blotting indicated 66.6% less SOX9 (BEC marker, CI: 95%) and 76.0% more osteopontin (inflammatory mediator produced by BEC, CI: 95%) in KO mice with BDLs than in WTs. Interestingly, there was also 96.0% more osteopontin (CI: 93%) in resting KO mouse livers compared to WT, suggesting a regulatory role for inflammation in bile duct proliferation/transdifferentiation. The combined results implicate a role for LRP1 in biliary regeneration following injury as well as normal biliary tubulogenesis.

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5. Determination of the Presence of Genetically Modified Indicator Genes in Carrot and Corn Samples Jennifer Miller, Dr. Marietta Wright Waynesburg University, Waynesburg, PA The presence of genetically modified organisms (GMOs) is occurring frequently in our society. The most common means of determining if an organism is genetically modified is to test for the Cauliflower Mosaic Virus (CaMV) promoter and the Argobacterium tumefacien (T-NOS) terminator gene indicator sequences. The purpose of this study was to determine if certain food items contained genetically modified indicator genes. Five different sample types were tested: organic labeled carrots, regular unlabeled carrots, unknown labeled carrots, unlabeled corn seeds, and regular unlabeled corn. DNA was extracted from each sample and amplified via the Polymerase Chain Reaction (PCR) for the GMO indicator genes. Two percent agarose gel electrophoresis was utilized to view the presence of the GMO genes of the different PCR products. Both indicator genes were observed only in the regular unlabeled corn sample: 160.44 base pairs (bp) for the CaMV, with an 80 percent certainty, and at 62.22 bp for the T-NOS, with a 49 percent certainty. Proper labeling of GMOs in food products is essential to personal dietary knowledge. While not much is known about the effects of genetically modified food on the human body, it is important for food products to be labeled properly. Therefore, future studies need to be conducted to determine how GMOs affect the human body.

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6. A pilot preclinical study to determine possible synergy between cisplatin and withaferin A in breast cancer cells Diana Mansour, Anuradha Sehrawat, Shivendra V Singh Molecular Pharmacology, SURP, University of Pittsburgh, Pittsburgh, PA Triple negative breast cancer patients treated with cisplatin not only experience severe side effects, including nephrotoxicity, hepatotoxicity, and myelosuppression, but also frequently become resistant to the drug. Development of potential combination therapy of cisplatin with naturally derived agents may attenuate these complications. A crystalline steroidal lactone isolated from the leaves and roots of Withania somnifera, Withaferin A (WA), is suggested as a potential anti-cancer compound and has been shown to prevent tumor growth, angiogenesis, and metastasis. Both in vitro and in vivo studies with WA have documented its pro-apoptotic and anti-tumor activity in breast cancer. In the present study, we explored the combined effect of WA and Withania somnifera root extract (WRE) with cisplatin for the treatment of triple negative breast cancer SUM159 cells. Trypan blue analysis facilitated cell viability analysis, whereas Annexin V FITC assay yielded apoptosis statistics. WA and WRE (0.25-1 µM) treatment caused dose-dependent decrease in viability of SUM159 cells in association with apoptosis induction. Compared to vehicle-treated control, SUM159 cells treated with different concentrations of cisplatin (5-20 µM) showed significant decreases in cell viability. However co-treatment of cells with 5 µM cisplatin and different concentrations of WA or WRE (0.1-1 µM) failed to elicit an additive effect on cell viability and apoptosis induction in SUM159 cells. From our results, we concluded that WA and WRE do not function synergistically with cisplatin for the treatment of SUM159 breast cancer cells.

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7. Analysis of Poly(2-oxazoline)s to control surgical blood loss

Kerry McGowen, Marcel Boerman, Jan Van Hest Radboud University Nijmegen, Institute for Molecules and Materials, The Netherlands Surgical wounds on soft tissue organs, such as the liver and kidneys, can lead to excessive blood loss and increased risk of infection and inflammation. Recent research suggests that hemostatic polymers are a promising alternative to traditional methods for repairing surgical wounds, such as sutures, staples, adhesive tapes, and collagen pads. Unlike these traditional methods, hemostatic polymers are mechanistically independent of the natural blood coagulation cascade and therefore exhibit quicker coagulation times. In this study, the ideal composition of the NHS-ester groups on the polymer, poly(2-oxazoline)s (POx), to function as a hemostatic agent was investigated. The NHS-ester groups serve to bind with amines present in blood protein creating a hydrogel barrier, which slows bleeding. NMR analysis was used to determine the percentage of functional groups during each stage of synthesis of the polymer. Shearing tests, GPC, and SEM results suggest that the synthesized Pox polymer is more effective in coagulating blood and forming a stronger bond than a collagen pad alone.

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8. Extracellular Ca2+ Required for Xenopus laevis Development Danielle M. Berlin, David J. Leftwich, and Anne E. Carlson, Ph.D. University of Pittsburgh, Pittsburgh, PA A rise in intracellular Ca2+ is a critical process in the fertilization and development of most animals. Although it has been well established that Ca2+ is released from intracellular stores in the egg, the role of extracellular Ca2+ has thus far been unclear. Results from a previous study indicate that extracellular Ca2+ is not critical for the development of the African Clawed Frog, Xenopus laevis. However, preliminary data have indicated that extracellular Ca2+ may in fact play an important role. In this study, the requirement of extracellular Ca2+ in the fertilization and development of Xenopus laevis was investigated through the use of the divalent chelators EGTA and EDTA. EGTA and EDTA were used to remove extracellular Ca2+, and development was assessed on the basis of cleavage furrows. Removal of extracellular Ca2+ with 100µM of chelator was found to halt development in almost 80% of embryos, while the addition of Ca2+ in the presence of chelator resulted in recovery of development. Varying concentrations of EGTA and EDTA were used to determine the minimum concentration of chelator needed to halt development. Binding affinities (KD) were determined for each chelator. The KD for EDTA was 3.79µM, and the KD for EGTA was 14.6µM. These results indicate that extracellular Ca2+ plays a critical role in Xenopus fertilization and development. This knowledge could help to elucidate the specific function of extracellular Ca2+ in development, and could potentially be used to develop novel contraceptives and address problems of infertility.

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9. Stingray Epidermal Mucus-associated Bacteria as Novel Sources of Antibiotics Derrion May1, Carl Luer2, Cathy Walsh2, and Kim Ritchie2 1Waynesburg University, Waynesburg, PA, 2Mote Marine Laboratory, Sarasota, FL Resistance to antibiotics has been a major cause of the increase in hospital and community associated infections over the past few decades. Because of the lack of new antibiotic discoveries, novel sources of bioactive compounds are being investigated. Elasmobranchs, such as stingrays and skates, have a strong innate immune system and have beneficial bacteria located on their epidermal surfaces. These bacteria may act as a first line of defense for elasmobranchs and provide a novel source of antibiotics for drug discovery. The purpose of this experiment is to investigate antibiotic-producing bacteria from the cownose ray, Rhinoptera bonasus, and the devil ray, Mobula hypostoma, for their ability to continue producing antibiotics when grown in larger batch cultures. If these bacteria continue to inhibit growth of MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin- resistant Enterococcus) after scale-up, they may serve as a feasible source of antibiotics for drug discovery.

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10. The Effects of Smoking on Cardiorespiratory Fitness in College Students Steffen Simerlink, Kristianna Bancroft, Sabbath Friend, and Cody Kuhel Washington and Jefferson College, Washington, PA Smoking cigarettes, marijuana, or a combination of both has been suggested to damage the respiratory system and hinder cardiorespiratory fitness. Few studies have evaluated the effect of smoking cigarettes or marijuana on a physically active college-aged sample. The purpose of this study was to assess the effects of smoking cigarettes, marijuana, or a combination of both, on VO2max in both trained and untrained college-aged individuals. Participants were subject to a Modified Bruce Treadmill Test. Throughout the test, heart rate was monitored and respiratory gases were collected in Douglas bags. Gas analysis was completed on the expired respiratory gases to calculate VO2max. Twenty nonsmokers (19 trained, 1 untrained) and 11 smokers (9 trained, 2 untrained) were studied. Smokers had a higher systolic blood pressure before exercise (130 ± 11 mm Hg) than nonsmokers (118 ± 13 mm Hg, p = 0.021). Smokers also had higher heart rates during the recovery period than nonsmokers at 0, 45, 90, 135, and 180 seconds after termination of exercise (p = 0. 033, p = 0.092, p = 0.032, p = 0.033, p = 0.161, respectively). Irrespective of physical activity level, nonsmokers exhibited a slightly higher relative VO2max (3.57 ± 2.11 mL kg-1 min-1) than smokers (3.56 ± 1.84 mL kg-1 min-1, p = 0.992). Trained smokers had a slightly higher relative VO2max (3.89 ± 1.87 mL kg-1 min-1) than trained nonsmokers (3.62 ± 2.15 mL kg-1 min-1, p = 0.756). In contrast, untrained smokers had a slightly lower relative VO2max (2.08 ± 0.42 mL kg-1 min-1) than the untrained nonsmoker (2.64 mL kg-1 min-1). These data suggest that smoking’s effects on the respiratory system haven’t taken effect yet because the smoking habits haven’t persisted for a long enough period. However, the effects of smoking on the cardiovascular system appear to have been established despite the brief smoking history of the participants.

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11. Does Sand, or Sawdust Bedding Produce More Visible Abrasions on Bos taurus (Dairy Cattle)? Julianne Holler Thiel College, Greenville, Pennsylvania The abrasiveness of sand versus sawdust bedding on the teats of Bos taurus, dairy cattle was studied on 16 dairy cows. Previous studies suggested that sand bedding would be more abrasive on the teats than would sawdust bedding because sand is coarser. Therefore, sand bedding was expected to cause more cuts and scratches on the teats of cattle than sawdust. All cattle were originally on sawdust bedding; at the start of the experiment a group of 8 remained on sawdust, while another 8 were put on sand. After 3 days, these two groups were switched. It was observed that the sand was slightly more abrasive than sawdust bedding. The result was weakly significant. These findings support the initial hypothesis, but greater sample sized of cattle would be needed to confirm the conclusion.

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12. Bryophyte and sediment bacterial communities from streams in Southwestern PA Aric Brown, Chris Casagrande, Flaviane Nunes da Silva, and Luisa Lopes de Souza Department of Biology, Washington & Jefferson College, Washington, PA Headwater streams are the location of many important ecological processes such as biogeochemical cycling but are often neglected in conservation efforts. There is currently a need to monitor the effects of anthropological stressors such as hydraulic fracturing and mining on these streams. Bacteria have been shown to be good indicators of environmental health. Because of this, this study aimed to characterize the sediment and bryophyte bacteria in two headwater streams at Abernathy Field Station (AFS), a mixed-forest research site in Southwestern Pennsylvania that is proximal to different stressors. The samples were collected from two streams, Jefferson’s Run (groundwater fed) and Abe’s Creek (surface water fed), at AFS in winter 2015. The 16S ribosomal DNA gene, a phylogenetically significant marker with which bacteria can be identified, was amplified from the samples. 16S rDNA was isolated with a culture-independent method and then cloned into E.coli. Subsequently, the E.coli library was screened and the 16S rDNA was sequenced from 279 clones (138 sediment and 141 bryophyte). Ribosomal DNA sequences were classified and analyzed using the Ribosomal Database Project and Mothur, respectively. The bacteria were grouped into 14 different phyla. Interestingly, although the two streams were different in size and water flow, their bacterial communities in the streams did not substantially differ in phylum distribution. The bacterial community in the combined sediment samples was found to be more diverse than that of the combined bryophyte samples. While further work still needs to be done, this study positively contributed to the monitoring of AFS streams.

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13. Increased Mobility of Captive Penguins through Olfactory System Enrichment Amanda Cavender Thiel College, Greenville, PA Penguins rely heavily on their eyesight to find food in the wild, but when they age their eyesight begins to deteriorate. Many zoo keepers are trying to find ways to stop older penguins from losing mobility and their desire to eat when their vision begins to diminish. I wanted to see if the captive penguins at the zoo would be able to learn a new method of finding their food in the water with only their sense of smell. I created a device with a buoy, plastic chain, fake kelp, and a weight to hide Capelin, the penguin’s food fish, underwater. I predicted that, after repeated exposure to the device, the penguins would be more likely to find the hidden fish. After eleven trials spanned through six months, I found that there was a slight increase in the number of fish that were found, pulled, and eaten off of the chain. Many of the older penguins did not jump into the water, so it was hard to test if they would have learned to use their sense of smell. The younger penguins, however, were very involved and eager to learn the new method of catching food. The test lead me to believe that with longer exposure to the device, the penguins would learn to find their fish more so with their sense of smell.

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14. Population Genetics of Posthodiplostomum minimum in East Central Ohio Andrya Durr and Wayne Rossiter In the field of parasite ecology, there is continued interest in defining the relationship between parasite and host population dynamics over geographic space. This study focuses on the prevalence and genetic diversity of Posthodiplistomum minimum in host sunfishes (Lepomis spp.) across several lakes within a 60-mile radius. Sunfishes are intermediate hosts for the parasite, where the metacercariae will encyst predominantly in the heart and liver. Sunfishes were collected from multiple lakes of differing size (from 5 to 2,500 acres), all located in east central Ohio, and dissected for the extraction of cysts. Cysts were pooled from each individual host, and DNA was extracted for amplification of the COI barcode via polymerase chain reaction (PCR). Sequences were added to existing published data for this parasite, and were incorporated into a phylogeny of the genus Posthodiplostomum using MEGA2. Additionally, we produced haplotype trees to examine genetic differences (and connectivity) among sites. Our results suggest differences in infection prevalence and intensity among different host species. Tracking these parasites and their evolution is important in understanding their life cycles, as well as how they adapt to their environment and surroundings. Also, verifying differences in host susceptibility and parasite genetics could lead to future studies regarding parasite-host evolution.

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15. Altered Expression of the Multifunctional Sorting Protein PACS-2 Correlates with Changes in Cellular Response to Hypoxia Benjamin Mayro, Jonathan Barroso, Laurel Thomas, Satoshi Nakajima, Gary Thomas. Molecular Virology and Microbiology, SURP University of Pittsburgh, Pennsylvania Solid tumor growth creates an intratumoral hypoxic environment that slows tumor progression. Cancer cells respond to the hypoxic environment by inducing the expression of hypoxia inducible factor-α (HIF-1α), which is a transcription factor that induces expression of target genes, including VEGF, which attracts new blood supply. Under normoxic conditions, hydroxylated HIF-1α is targeted for degradation by the von Hippel-Lindau tumor suppressor (VHL), which is part of an E3-ubiquitin ligase complex. VHL stability and functional ability is regulated by posttranslational modifications and protein-protein interactions. Interestingly, VHL has an acidic cluster that is characteristic of a binding motif for the multifunctional sorting protein PACS-2. We therefore asked whether PACS-2 may modulate the HIF-1α-VEGF angiogenic axis. Co-immunoprecipitation analysis showed PACS-2 interacts with VHL, suggesting PACS-2 may modulate HIF-1α induction. To test this possibility, PACS-2 was knocked out in HeLa cells using CRISPR/Cas9 technology. Hypoxia was induced in Hela WT and PACS-2KO cells by the addition of cobalt chloride (150 µM) for 24 hours. We observed that loss of PACS-2 lead to a marked increase in HIF-1α expression during hypoxia. Further, increased HIF-1α expression in WT cells during hypoxia was coupled with decreased PACS-2 expression. Together, these results suggest PACS-2 supports VHL-mediated degradation of HIF- 1α. The interaction between the PACS-2 and VHL provides a prospective mechanism for PACS- 2 involvement in hypoxia.

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16. Internship at Microbac Laboratories, Inc.

Jonecca Robinson Microbac Laboratories, Inc. Sayre, PA The internship was completed at Microbac Laboratories, Inc. in Sayre, PA. Microbac is an environmental laboratory that specializes in drinking and waste water testing. The internship was completed in the Microbiology and Sample Receiving Departments. Tasks completed in Microbiology were to test drinking water for total coliform and e. coli. Positive total coliform tests changed to a yellow color and positive e. coli tests fluoresced under UV light at 254 nm wavelength. The time spent in sample receiving was spent using ELEMENT, which was the laboratory information management system for Microbac. ELEMENT is the program that allows for paper chain of custodies to be turned into digital copies and sample labels made. Bottle orders were made in sample receiving for the repeat customer orders in order to save time, travel, and money. The internship reinforced the importance of stewardship, sample integrity, lab safety, quality control and quality assurance, and cleanliness of the testing area. The internship was very beneficial and helped reinforce many of the concepts that Seton Hill has in the curriculum.

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17. Lambda Red-mediated recombination of core gene clusters in systemic Salmonella enterica subsp. enterica serovars Aric Brown, Min Yue, Dieter Schifferli School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA Salmonella enterica subsp. enterica is well-characterized as causing enteritis in humans and other animals. Certain serovars of S. enterica subsp. enterica, however, are also capable of causing more serious systemic infections (i.e. sepsis). The ability of some serovars to cause systemic infection while others cannot is not yet fully understood. Through previous metagenomic analyses, unique gene clusters have been identified in systemic serovars that are absent in intestinal serovars. The aim of this study was to create mutants of S. enterica subsp. enterica systemic serovars lacking these gene clusters as a way to identify and characterize the genes suspected to be responsible for the ability of certain serovars to cause systemic infections. In this study, Lamba Red-recombination was used to create the mutants. Through Lambda Red-recombination, the genes of interest were replaced with an antibiotic cassette, which was used to select for the mutants. Specifically, two genes, tentatively called yjcS and tdt in this study, were replaced with a kanamycin cassette in Salmonella enterica subsp. enterica serovar Dublin, a systemic serovar. Phenotypic assays can now be performed to compare the wildtype and mutant serovars as a way to characterize the target genes. Future studies should create other systemic Salmonella serovar mutants lacking the same genes and use an expression vector to express the genes in nonpathogenic E. coli and intestinal Salmonella serovars to further explore gene function. By gaining a better understanding of the suspect genes, new therapies for systemic Salmonella infections can potentially be developed.

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18. Determining the Mechanism of Neutrophil Killing During Listeria monocytogenes Infection Catherine McCormick, Cheyanne Fedder, and Delbert Abi Abdallah Thiel College, Greenville, PA 16125 Listeria monocytogenes is a gram positive bacterium that causes listeriosis infections in humans. L. monocytogenes is a facultative intracellular bacterial pathogen that tightly regulates activities of various virulence factors during infection. A mutant strain, that has lost the ability to control the activity of phospholipase C (PC-PLC), is attenuated during infections. Given that neutrophil cells are the first to arrive at a site of infection, we sought to elucidate whether knock-outs are more susceptible to killing by neutrophils. Additionally, we sought to elucidate the specific killing mechanisms employed by those cells. Data collected to date show that mutants are more susceptible to killing by neutrophils and that the intracellular protein myeloperoxidase (MPO) is downregulated by the wild-type strain.

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19. Determining the Effectiveness of Pure Plant Extracts on inhibiting Clostridium sporogenes growth as Compared to Certain Antimicrobial Drug Classes Arien Skiba and Delbert Abi Abdallah Thiel College, Greenville, PA 16125 Clostridium sporogenes is a gram-positive, streptococcal bacillus, obligate anaerobic bacterium which is motile by Peritrichous flagella. C. sporogenes shares a similar ecology to Clostridium botulinum, a common, virulent bacterium of the gastrointestinal tract (GI) of dogs, but does not produce the endotoxin that C. botulinum produces. C. sporogenes was grown for that very reason. This study sought to investigate the bacterial inhibitory qualities of several plant extracts and compare their effectiveness to certain class drugs. The study sought, also, to compare the inhibitory effectiveness between two, separate species of Oregano. Experiments are currently ongoing and concrete analysis of the data will be presented at the conference.

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20. Bioinformatics: Annotation of the Mycobacteriophage Pipsqueak Using DNA Master and Phamerator Hannah Budzowski Thiel College, Greenville, PA Bioinformatics is used to analyze novel DNA sequences and genes in newly discovered organisms, such as bacteriophages, viruses, bacteria, and animals. One newly discovered mycobacteriophage is Pipsqueak, which was discovered by the Champlin High School Microbiology Club in 2011. Currently, students at Thiel College are fully analyzing and annotating the genome. My project uses two bioinformatics programs DNA Master and Phamerator to annotate base pairs 35,000 – 50,000 of the mycobacteriophage named Pipsqueak. DNA Master and Phamerator are bioinformatics tools that enabled me to find several important aspects of each gene including the start and stop codons, as well as BLAST analysis which aligns the sequence with other mycobacteriophage genes. One interesting Pipsqueak gene that I analyzed was gene 56, which had a high sequence alignment with RepA, replication protein A, from E. coli. This sequence was also found in several other mycobacteriophages but in E.coli RepA encodes for a helicase. With the help of several Thiel College students, Pipsqueak will soon be completely annotated and submitted to GenBank. This will allow it to be accessible to students, teachers, and biologists around the world.

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21. Evaluation of Luminol, Bluestar, and Hemascein and their Effectiveness on Cleaned Up Bloodstains Chelsea Smetanka Seton Hill University, Greensburg, PA Luminol, Bluestar, and Hemascein are three presumptive test used to identify possible bloodstains. In this project, these tests were evaluated to determine their effectiveness on cleaned up bloodstains. Human blood was pipetted onto cotton and denim swatches and allowed to dry for various times. The amount of blood used per swatch was 100µL. The drying times evaluated were 30 minutes, 4 hours, 2 days, 1 week, and 2 weeks. After the appropriate drying time, the stains were ‘cleaned up’ using paper towels and 12 various cleaning products. The cleaning products used were Tide to Go sticks, Stain Devil stain remover, shout, Dawn, generic dish soap, Wisk detergent, Tide detergent, OxyClean detergent, hydrogen peroxide, vinegar, ammonia, and corn starch. Three trials for each material, cleaning product, and drying time were analyzed. The samples were sprayed with the appropriate presumptive test and the luminescence of each stain was observed. All three tests were able to detect the presence of blood on all swatches by luminescing. Only photographs of Bluestar luminescence could be obtained. Hemascein was more difficult to use and the luminescence was harder to observe than Luminol and Bluestar. Based on the results, Bluestar seemed to be the most effective on detecting cleaned up bloodstains.

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22. Characterization of a novel Pedobacter strain from petunia rhizosphere. Kayla Parr, Daryl R. Sas, PhD and David A. Essig, PhD Department of Biology, Geneva College, Beaver Falls, PA 15010. The Pedobacter genus is a relatively new genus and was defined by the heparin degrading species Pedobacter heparinus. Members of the genus include over 35 recognized species which are Gram-stain negative, strictly aerobic rods, with or without gliding motility and genomic DNA %GC of 36 – 45%. Pedobacter strains have been found in diverse environments including soil, compost, glacier criconite and fresh water streams and ponds. In this study, strain G11 was isolated from the rhizosphere (root adherent fraction) of a petunia plant growing in northeastern Ohio. 16S rRNA gene sequence analysis revealed that strain G11 belonged to the genus Pedobacter (94-99% similarity). Strain G11 produces a pink pigment on TSA plates with colonies that are circular, raised with entire margins and 1 mm in diameter. The strain was Gram-stain negative and consisted of short bacilli in diplo arrangement. Other assays indicated that the strain was positive for oxidase and catalase, but was negative for nitrate reduction, growth on MacConkey agar and gliding motility. The temperature for optimum growth was 25 oC, with no growth at 4 or 37 oC. Results indicated that G11 was distinguishable from the most related Pedobacter species (agri and terrae) by a number of phenotypic characteristics. Our preliminary findings suggest that G11 is a potentially novel root associated Pedobacter species.

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23. Anxiety QTL found on mouse chromosome 1 using consomic strains and plus maze. Ken Kutcel, Charlotte Glasstetter, Jordan Baum, Matthew McHugh and David Essig, PhD Department of Biology, Geneva College, Beaver Falls, PA 15010 Anxiety disorders are a common mental condition (~15% of population in the US) and are due to a combination of predisposing genes and environmental factors. Singer et al. (2005) used consomic or chromosome substitution strains (CSS) involving the low anxiety host strain (C57BL/6J) substituted with chromosomes from the high anxiety (A/J) strain and found quantitative trait loci (QTL) for anxiety on chromosomes 1 and 6. These investigators used the open field and light-dark box to induce anxiety behavior. However, there are established differences between these anxiety producing tasks and the more commonly used plus maze task. Moreover, the latter is often used in testing candidate anxiolytic drugs for humans. Experiments were performed with A/J, C57BL/6J and CSS-1 mouse strains (n = 10 each) and tests conducted using the plus maze. Several measures indicated a significantly (p < 0.05) higher level of anxiety in CSS-1, as compared to C-57 mice: CSS-1 mice produced more fecal boli than C-57 (C-57: 2.09±.44, A/J: 4.33±2.40, CSS-1: 3.55±.56). A significantly (p < 0.05) greater percentage of CSS-1’s arm crosses were into closed arms compared to C-57 (C-57: 58.5±.9%, A/J: 74.3±4.1%, CSS-1: 73.5±1.0%). CSS-1 had less (p < 0.05) total crosses than C-57 (C-57: 26.6±2.9, A/J: 2.1±.5, CSS-1: 14.1±1.4). We conclude that chromosome 1 harbors at least one QTL which is an important genetic determinant of anxiety produced during several tests including the plus maze.

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24. Annotation of putative polysaccharide utilization loci (PUL’s) in Pedobacter heparinus Sarah Elliott, Ken Kutcel, Matt Banyas, Emily Neice, Stevie Byers, Catherine Simmons and David A. Essig, PhD Department of Biology, Geneva College, Beaver Falls, PA 15010. Pedobacter heparinus is the type-species for the relatively new Pedobacter genus which now contains over 35 member species. The permanent draft genome for P. heparinus has ~4,500 protein coding genes, 65% of which have predicted products and function. However, annotation which enumerates the interactions between gene products, especially those within operons or regulons has not been performed for this type-species and genus. One notable feature of genomes within this genus is the abundance genes coding for homologs of SusC and SusD like proteins. SusC is an oligosaccharide transporter and SusD is involved in binding to a specific extracellular polysaccharide. In P. heparinus there are over 50 SusC/SusD gene pairs with the SusD gene located immediately downstream of the SusC gene. Our project focused on the bioinformatic characterization of genes/operons surrounding the SusC and D gene pairs in two genomic regions. In locus 1 (Phep 1149 to 1162), SusC and D genes were flanked by genes whose predicted products included pectin degradation enzymes (periplasmic pectin lyases, pectinesteraes, and glycoside hydrolases). Genes coding for an inner membrane transporter and a Lac I repressor like protein were also found. In locus 2 (Phep 0954 to 0966), SusC and D genes were flanked by genes whose predicted products included a sigma factor, anti-sigma factor, pectin or heparin lyases, and several glycoside hydrolases. Our annotation reveals two putative polysaacharide loci with over 25 genes likely involved in extracellular pectin sensing/binding, transcription regulation, pectin degradation and transport of monosaccharide byproducts.

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25. The Creation of a Double Knockout to Test the Mutator Effect of the dgt gene in E. coli. Morgan Murphy and Steve Kozuch Thiel College, Greenville, Pennsylvania The dgt gene in Escherichia coli (E.coli) is responsible for the production of dGTPase, which has been demonstrated to catalyze the hydrolysis of dGTP, thereby producing deoxyguanosine and triphosphate. As a result, this regulates the amount of dGTP available in the cell which helps keep mutations low in the bacterium. When the dgt gene is inactivated, this enzyme is not functional and the number of spontaneous mutations increase. The objective of this project is to investigate other genes within E. coli’s genome to determine if they collaborate with the dgt gene to regulate spontaneous mutations via a double knockout. Mutated E. coli strains, with single gene knockouts, were provided by GenoBase from the “Keio Collection.” The research conducted utilizes P1 phage lysates created from the six Keio strains including: ada, cadC, dsdC, nhaR, soxR, and uxuR. A double knockout was created using these P1 lysates via transduction and was then measured by a papillation assay to quantify the mutator effect in E. coli. Studying the inactivated dgt gene in E. coli along with other inactivated genes, our results suggest some of these genes may be involved in regulating spontaneous mutations. We conclude that genes, such as nhaR, cadC, and soxR, in E. coli collaborate with the dgt gene to regulate spontaneous mutations. Collaboration of these genes with the dgt gene can add knowledge to basic research and further lead to discoveries in more advanced organisms.

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26. Role of Rab22 in ENaC trafficking through early and recycling endosomes in the presence of aldosterone Abigail Guest, Victoria Kissell Lindsey Dove, and Stephanie Solida Washington and Jefferson College, Washington, PA Control of epithelial sodium channel (ENaC) trafficking by hormone level can be demonstrated in the cortical collecting duct cells of the mouse kidney (mCCD), a well described native model of aldosterone sensitive transport tissue. Aldosterone’s role in trafficking seems to influence the involvement of a family of Rab GTPases, namely the Rab22. The current study is directed at the role of Rab22 in ENaC traffic through early and recycling endosomes; dominant active (DA) and dominant negative (DN) forms of Rab22 were synthesized and transiently expressed in mCCD cells. Expression was confirmed using confocal microscopy and the effects of wild-type, DA, and DN Rab22 were examined by short circuit current analysis, Ussing chamber studies. Confocal microscopy confirmed ENaC localization in subapical pools and biosynthesis throughout the endoplasmic reticulum in a diffuse pattern. Poor transfection efficiency, illustrated through confocal microscopy, could be the cause of insignificant differences in current seen across the four cell types during short circuit analysis studies. The addition of forkolin was inconsequential due to the lack of phenotypic expression and low number of trials (n=5). Though phenotype was poorly expressed in the cells, Na+ current was confirmed by amiloride addition and successfully recorded.

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27. Magnetotactic Bacteria as Symbionts of Geomagnetically Migratory Organisms Charles Cook, Dr. Chad Sethman Waynesburg University, Waynesburg PA Magnetotactic bacteria (MTB) are able to orient themselves and navigate within the earth’s geomagnetic field via a process known as magnetotaxis. Magnetotaxis is enabled by the possession of the magnetosome, which is a linear arrangement of magnetite crystals, akin to a compass needle, within an area of the bacterial cell’s cytoplasm. MTBs are ubiquitous in nature and are highly prevalent in the top layers of aquatic sediments, however the purpose of their magnetotaxis is currently unclear. Many geomagnetically responsive (migratory) organisms such as whales, fish, amphibians, birds, invertebrates etc. share the same environment with MTBs, however their biophysical mechanism of magnetoreception is also unclear. We hypothesize that the mechanism of magnetoreception used by these multicellular organisms involves harboring symbiotic MTBs within sensory organs. In order to look for the presence of MTBs we are developing a polymerase chain reaction (PCR)-based approach that will be able to detect the bacterial magnetosome-specific gene, mamB, in specimens of sensory tissue. The mamB gene has been shown to be essential for the formation of magnetosomes and is highly conserved among MTBs.

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28. Optimistic thinking and the immune system strength Mary Smith and Victoria Hogg Thiel College Greenville PA. This study investigated the immune strength of individuals and the effect their level of optimistic thinking during a stressful event had. This was done by asking Thiel College students that were enrolled in Cell Biology in the spring semester of 2016 to give saliva samples and fill out a survey. The surveys tested the participants’ optimism and the saliva samples were tested for the levels of the stress hormone, cortisol, to look for an immune response. Since many college students are very stressed about exams and studying for them, exams were used as the stressful event for the study. Samples were collected during two of the course’s exams and placed in a -80C environment until they were able to be tested. Two control samples were also taken randomly in the semester during non-stressful events. Once the samples were collected we performed an eliza test to test for the cortisol levels. The surveys were scored and the four samples and surveys given by each participant were compared to the other samples and surveys from that participant in order to investigate whether there was a change in optimism level for each individual during stressful events compared to non-stressful events. If changes in optimism levels were detected, changes in cortisol levels were looked for to see if there was any possible correlation between the two.

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29. Retrospective Evaluation of High Density Spinal Cord Stimulation Programming: Conversions, Trials and Implants Cody Kuhel, David Provenzano M.D. Pain Diagnostics and Interventional Care, Sewickley, PA OBJECTIVE: The primary objective is to determine the efficacy and safety of high density (HD) sub-perception spinal cord stimulation (SCS) programming. METHODS: Following IRB approval, a retrospective review was performed on SCS patients whom underwent HD programming. For HD programming, all amplitude settings were below paresthesia threshold. The three HD categories included trial, implant, and attempted conversion from conventional to HD. RESULTS: 16 patients were identified including 12 conversion patients and 4 patients who were had positive response to HD programming during the trial and then were implanted (Table 1). The average programing parameters for the conventional group were 2.9 ± 2.1 mA, 447.9 ± 203.7 µs, 84.85 ± 98.4 Hz and a pulse density of 3.25% ± 2.9%. Average programming parameters for the HD group were 2.1 ± 1.7 mA, 506.2 ± 297.35 µs, 657.1 ± 378.9 (Hz), and a pulse density of 22.9% ± 2.4%. Figures 1 and 2 demonstrate programming preference and pain scores. Following implant, only 2 of the 4 patients continued to prefer HD. For individuals that preferred HD, the average time of use was 23.6 weeks. Conclusion: Conventional and HD programming patients reported a reduction in pain scores. In subpopulations, HD programming maybe a viable option to deliver subthreshold pain reduction. Although in patients with attempted conversions, a majority of patients preferred conventional programming and in the implanted patients only 50% of tested patients continued to prefer HD programming. Investigation through prospective studies is warranted.

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30. Internship at the Westmoreland County Coroner’s Office Mikayla Beranek Westmoreland County Coroner’s Office, Greensburg, PA This internship was performed at the Westmoreland County Coroner’s Office in Greensburg, Pennsylvania. The responsibilities of the coroner’s office include performing investigations to determine cause and manner of death as well as deciding whether a death may have resulted from a criminal act. Throughout the duration of this internship, there were four crime scene investigations and one autopsy that were observed. Two of the four decedents from the crime scenes were suicides caused by asphyxiation due to ligature hanging. The other two decedents were thought to have died from a possible drug overdose. Since the cause and manner of death could not be determined at those two crime scenes, an autopsy would be performed and a toxicology sample would be sent to NMS labs. The other technique observed was an autopsy procedure performed on a woman that was thought to have died from a possible drug overdose. The grooves in her brain indicated that the she might have put drugs into her body so a toxicology sample was sent to NMS labs.

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31. Mitochondrial DNA Sequencing for the Determination of Family Lineage Harbinder Jawanda Seton Hill University, Greensburg, PA. Human Mitochondrial DNA (mtDNA) has previously been studied to understand genetic lineages or mutations, in both coding and noncoding regions. DNA sequencing is a biotechnology technique used to determine the order of the four-nucleotide bases that comprise a DNA strand. Several methods have been developed for the experiment of sequencing DNA such as Primer Walking and Shotgun Sequencing. Here we propose an approach to identify familial inheritance of mtDNA by extracting, purifying and amplifying mtDNA followed by Sanger Sequecning using an Applied Biosystems 310 genetic analyzer. This approach involves a multi-step process. The DNA was firstly extracted from the swab samples obtained from family saliva and amplified using a Forward Primer and a Reverse Primer. DNA was separated for amplification by gel electrophoresis and purified using a Terminator BigDye kit. Prior to DNA sequencing, the samples attained were re-amplified using the same Terminator BigDye kit. The resultant amplicon strands containing dyes corresponding to different nucleotide bases in the DNA fragments are identified by activation when passed through a laser beam from the 310 Genetic Anlyzer. Resultant data showing DNA sequence data is displayed on a screen for analysis. No sequencing data was attained from the 310 despite the fact an expected fragment was observed on a gel electrophoresis. This result can be explained by sensitivity of 310 Genetic Analyzer. Additionally, contamination and loss of product through out the multiple steps may lead to distorted results.

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32. Avoidance Response of C elegans OSM-9, EGL-30 and EAT-16 knockouts to volatile odorants Octanol and Octanone Kristin Davis, (He Liu, Ph.D.), Gannon University- Biology This experiment was intended to discern the role of OSM-9, EGL-30, and EAT-16, in the response of C elegans to the volatile odorants octanol and octanone. In the ASH neuron of C elegans, OSM-9 encodes a TRPV channel that is used to sense osmotic pressure. EGL-30 is Gqα protein and acts upstream of OSM-9 to regulate ASH depolarization in response to high osmolarity. EAT-16 is a RGS protein to inhibit the functions of EGL-30. In our behavioral tests, platinum wire was dipped in octanol or octanone and placed just in front of an animal moving forward. The time it took each animal reverse direction was measured. The OSM-9 knockout was the only strain to show a significant delay in response time, being at least 8-fold slower on average than the wild type worms. EGL-30 and EAT-16 knockouts did not exhibit a significantly different response time. Our results indicate that the TRPV channel gene OSM-9, but not the regulatory pathway through EGL-30 and EAT-16, is involved in the regulation of the avoidance response of C elegans to volatile odorants.