2016 Cynthia B. Peterson Poster Competition Abstract booklet

2nd Annual Cynthia B. Peterson Poster Competition Hosted by the Program for Excellence and Equity in Research

UTK ORNL GST/BCMB Retreat March 4, 2016

TABLE OF CONTENTS Dr. Cynthia Peterson: A Peek at PEER……….…................………Page 4 2015 Poster Competition Winners…………………………………Page 5 Genome Science & Technology Abstracts………………….……..Page 6 Biochemistry & Cellular and Molecular Biology Abstracts............Page 15 Chemical and Biomolecular Engineering Abstracts……………...Page 25 Chemistry Abstracts ………………….…………………………..Page 25 Comparative and Experimental Medicine Abstracts……………...Page 27 Ecology & Evolutionary Biology Abstracts……………..……….Page 27 Microbiology Abstracts………………….…………...….……….Page 29 Public Health Abstracts…………………….…………………….Page 29 2016 Poster Competition Judges…………………………..……...Page 30

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Dr. Cynthia Peterson A Peek at PEER Dr. Cynthia Peterson, who previously served as professor and Head of Biochemistry & Cellular and Molecular Biology (BCMB), was the initial principal investigator for The Program for Excellence & Equity in Research (PEER). PEER is an initiative to increase the number of exceptional underrepresented students graduating with doctoral degrees in STEM disciplines at the University of Tennessee, Knoxville. PEER seeks to advance a doctoral student’s competitiveness and interest in their chosen career field by offering professional development, ambitious research and dynamic mentoring opportunities. During her career, she has expanded her roles beyond a researcher and educator to include academic leadership and innovation. She was widely involved in UTK committees and strategic planning. As a woman in a field underrepresented with women, she brought to PEER a sensitivity to challenges that focused on an underrepresented individual’s successful pursuit of a Ph.D. and research career. Dr. Peterson is an internationally recognized researcher who studies the interactions among circulatory proteins and their roles in regulating hemostasis, the inflammatory response, infectious disease, and the biological clock.

Her research integrates biochemistry, biophysics, and molecular, cellular, computational, and structural biology. Dr. Peterson currently serves as College of Science Dean at Louisiana State University.

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2015 Poster Competition Winners 1st Place – Kristen Holbrook, BCMB (Advisor: Barry Bruce) 2nd Place – Arkadipta Bakshi, BCMB (Advisor: Brad Binder) 3rd Place (tie) – Ramya Enganti, GST (Albrecht Von Arnim) 3rd Place (tie) – Lindsey O’Neal, BCMB (Gladys Alexandre)

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2016 Cynthia B. Peterson STEM Poster Competition Abstracts (Departmental exhibition posters have been omitted.)

Genome Science & Technology Student Abstracts 1. Relating Dynamics and Function in AN Enzyme Family Khushboo Bafna*,1,2 and Pratul K. Agarwal1,2 1 Genome Science and Technology (GST) Program, University of Tennessee, Knoxville, Tennessee 2 Computational Biology Institute, Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee *Presenter: kbafna@vols.utk.edu Dynamics and structure play critical role in the function of biomolecules including enzymes. The role of structure in enzyme catalysis has been investigated for some time. However, only recently insights into the role of internal protein motions (protein dynamics) in enzyme catalysis have become available. Much like conservation of functionally important protein structural elements, characterizing similarities and differences in dynamics associated with catalysis by members of an enzyme family could provide vital information. The enzymes of the ribonuclease (RNase) family catalyze the hydrolysis of single stranded RNA molecules. They play a critical role in RNA degradation and defense against RNA viruses. Their mechanism and function are highly conserved within eukaryotes. In humans, there exist 8 canonical forms of Rnase possessing identical or similar active site residues and conserved fold architecture.

These enzyme members of this family preserve varying degree of ribonucleolytic activity but, their catalytic efficiency differ by 10-5–10-6 fold. Ability of protein function (like enzyme catalysis) is closely linked to the ability to sample conformational sub-states that promote specific interaction between reactants at various stages of catalysis. Using theoretical modeling and atomistic molecular simulations at microsecond time scale, we have investigated the role of functionally important conformational sub-states in relation to optimal catalysis by the members of the pancreatic Rnase enzyme family. A total of 21 apo enzymes, and 7 canonical human Rnases and bovine Rnase A each with two different substrates (ACAC and AUAU) have been characterized. Results indicate that the dynamics of distal regions (particularly surface loop regions) plays important role in catalysis by the members of family. Results indicate that even though sharing the same fold the primary dynamics of members is different. Similarity of dynamics is more related to the biological function rather than a higher degree of sequence similarity, providing new insights.

2. Identification of Potential Interacting Partners of the Arabidopsis thaliana ETHYLENE RESPONSE 1 and 2 (ETR1&2) Ethylene Receptor Important for Functional Divergence.

Arkadipta Bakshi1, Sarbottam Piya3, Tarek Hewezi1,3, Brad Binder1,2 1) UT-ORNL Graduate School of Genome Science and Technology.

2) Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee.

3) Department of Plant Sciences, University of Tennessee, Institute of Agriculture.

The gaseous plant hormone ethylene is a simple, unsaturated hydrocarbon, involved in the regulation of a number of physiological and developmental processes in higher plants including seed germination, growth, fruit ripening, abscission and responses to various stress factors. There are five ethylene receptor isoforms called ETHYLENE RESPONSE1 (ETR1), ETR2, ETHYLENE RESPONSE SENSOR1 (ERS1), ERS2 and ETHYLENE INSENSITIVE4 (EIN4) that mediate the responses to ethylene in the model plant Arabidopsis thaliana.

Prior research has shown that these five-ethylene receptor isoforms in Arabidopsis have both overlapping and nonoverlapping roles in regulating diverse responses. Interestingly, ETR1 and ETR2 have been found to have contrasting roles in the control of Arabidopsis seed germination during salt stress as well as ethylene-stimulated nutational bending. All these examples of ETR1 sub-functionalization require the receiver domain of ETR1. Recently, we have reported that ETR1 receiver domain has multiple functions where residues in the Îł-loop are important for germination on salt and abscisic acid signaling, whereas residues on the C-terminal end of the receiver domain are essential for ethylene-stimulated nutations. Using site-specific mutagenesis of E666 in the receiver domain of ETR1, we have found that the mutation results in a receptor that inhibits germination more than the wild-type receptor. This mutation does not produce a hyper-functional receptor for the other traits studied. It is currently unclear about the receptorprotein interactions mediated by this region of the receiver domain. However, based on prior receptor-protein interaction studies; some possibilities include other ethylene receptors, CTR1, EIN2, His-containing phosphotransfer proteins, and response regulator proteins. The central objective of this study is to explore the requirement of ETR1 receiver domain in more detail and to identify potential interacting partners of C-terminal of ETR1, ETR2, and the etr1-E666A mutant with other signaling pathways involved in the control of this functional divergence.

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3. Characterization of the field-evolved resistance mechanism to Bt corn in Spodoptera frugiperda Rahul Banerjee (rbanerje@utk.edu)1, Lucas Hietala1, Fangneng Huang2 and Juan Luis Jurat-Fuentes1 1 University of Tennessee, Knoxville, TN, 2Louisiana State University, Baton Rouge, LA Cry proteins synthesized by the bacterium Bacillus thuringiensis (Bt) are pore forming toxins which are either produced by transgenic plants (Bt crops) or included in spray formulations to control agricultural insect pests. The extensive adoption of Bt crops represents high pressure for selection of resistance in target insect pests. Spodoptera frugiperda is a voracious polyphagous pest of economically important crops including corn, cotton and rice.

Populations of S. frugiperda developed resistance to transgenic corn producing the Cry1Fa Bt insecticidal protein. In our previous research we have shown that resistance in the 456 strain of S. frugiperda developed from field-collected individuals from Puerto Rico is associated to reduced Cry1Fa and Cry1A binding and reduced expression of selected alkaline phosphatase genes. In this work we present our findings towards elucidating the mechanism responsible for resistance in the 456 strain as well as its existence in resistant S. frugiperda collected in the southeastern USA. Host range study of a lysogenic phage in a roseophage-roseobacter system and characterization of lysogens in affecting marine biogeochemistry

4. Applying Sequence Clustering to Facilitate Sample Comparisons in Metaproteomes Searched Against Different Metagenomes in Human Microbiome Research J. A. Blakeley-Ruiz1, W. Xiong2, Y. Song3, C. M. Fraser-Liggett3, R. Hettich2; 1Univ.

of Tennessee, Knoxville, TN, 2Oak Ridge Natl. Lab., Oak Ridge, TN, 3Univ. of Maryland Sch. Of Med., Baltimore, MD

Introduction The human gut microbiome is an intricate, dynamic, and poorly understood ecosystem that plays an important role in human health and disease. MS/MS based shotgun proteomics is a powerful tool for characterizing this ecosystem by identifying proteins from a precompiled list of translated open reading frames derived from the genome of the sample in question. The size and specificity of this search space affects the false discovery rate of the entire analysis. To control the search space of the microbiome samples, we utilize a list of open reading frames for each sample derived from its own metagenome. This confounds the ability to make across-sample comparisons. Here we detail a clustering approach to enable sequence identity and functional category comparisons across-samples.

Methods Microbial proteins were extracted from stool samples collected from two adult human Crohn’s disease patients (deidentified) on days 0, 30, and 180 after resection surgery. The microbial metagenomes of each sample was sequenced and assembled into a list of open reading frames. Each microbial proteome sample was digested into peptides using trypsin, and measured in duplicate using 2D-LC-MS/MS on a high performance LTQ-Orbitrap-Elite. Identified spectra for each sample were searched against their specific metagenome concatenated with a human genome to identify both human and microbial proteins. Amino acid sequences from identified proteins were clustered by percent sequence identity and function to determine protein overlap at varying resolutions.

Preliminary Data The amino acid sequences from the metagenomes of all six samples were concatenated and clustered by sequence identity. Each of these clusters can be considered a distinct protein group. We identified a median 240 human, 3,580 microbial, and 3,831 total distinct groups per sample. Although clustering by 100% sequence identity was sufficient to remove the sequence redundancy between samples, this did not permit comparisons across samples since it yielded no protein overlap across all 6 samples. Clustering at 90% sequence identity better permits comparisons, with 25% of the protein groups found across all 6 samples. 90% sequence identity, however, can be ambiguous with respect to protein function, depending on which amino acids are substituted. Hidden Markov Models are able to calculate the probability that an amino acid will switch to another amino acid when comparing two sequences. The Pfam database employs Hidden Markov Models to group proteins into families of proteins of similar function and structure. We used HMMER to match the identified proteins to protein families in the Pfam database, which then further groups protein families into clans. We identified a median of 1477 protein families and 325 clans per sample. At the protein family level, we had a similar protein overlap to that found at 90% identity with 33% of protein families found in all 6 samples, but greater confidence in the functional similarity between the sequences matched to the protein family. At the clan level, we improved on our protein overlap across samples to greater than 50%, reinforcing the idea that protein overlap increases at lower resolutions. By identifying the best way to cluster proteins identified across samples, we have enhanced the ability to use differential metaproteomics to provide functional information about the human microbiome across temporal dynamics.

Novel Aspect Overcoming the challenge of comparing proteomes searched against different genomes using sequence-clustering methods.

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5. The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro.

Jennifer Bourn1,2 and Maria Cekanova1,2,* 1 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee, 37996, USA 2 UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, Tennessee, 37996, USA * Corresponding Author: mcekanov@utk.edu Several types of cancer, including transitional cell carcinoma (TCC) overexpress several receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR), c-kit receptor, epidermal growth factor receptor (EGFR), as well as the vascular endothelial growth factor receptor (VEGFR). Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with high expression of RTKs. Cyclooxygenase-2 (COX-2) is highly expressed in bladder cancer as well other types of cancers and is one of the key proteins during tumorigenesis. In this study, we validated the effects of RTKIs, Masitinib ® (AB1010) and Axitinib on cell proliferation of human (UMUC-3 and T24) and canine (K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) bladder TCC cells. Using WB analysis, tested human and canine TCC cells, except T24 cells, expressed PDGFR and c-kit receptors. There was no detectable EGFR and VEGFR expressions in any of tested cells by WB analysis. The K9TCC#1Lillie and K9TCC#5Lilly cells had a high expression of COX-2, however K9TCC#2Dakota and human T24 had moderate expression of COX-2, and no COX-2 expression was detected in UMUC3 cells by WB analysis.

Both RTKIs, Masitinib® and Axitinib inhibited cell proliferation of the tested human and canine bladder TCC cells in a dose-dependent manner by MTS and apoptosis assays. Interestingly, both tested RTKIs, Masitinib ® and Axitinib increased COX-2 protein expressions in tested canine TCC cells. Combined treatment of RTKIs with COX-2 inhibitors decreased cell proliferation of tested TCC cells more effectively as either treatments alone. Our results indicate a possible role of COX-2 signaling pathway in developed RTKIs-resistance in bladder cancer cells in vitro.

Co-treatment of RTKIs with COX-2 inhibitors might indicate better clinical outcomes in treatments of patients diagnosed with bladder cancer.

6. Integration of Drupal/Tripal Based Web Database with Elasticsearch: Hardwood Genomics Database (HGD) as an example case Chen, M1; Henry, NL1; Guignon, V2; Staton, ME1 1 University of Tennessee, Knoxville, Tennessee, USA 2 Bioversity International, Montpellier, France Tripal1 is a suite of Drupal2 modules which incorporates the community standard GMOD Chado 3 schema into a Drupal-based web database, providing a simplified solution to construction of informative, research-oriented websites for the biological scientific community. The number of Tripal-based websites have been increasing since the release of Tripal’s first version with a focus on disseminating genetic and genomic data for plant species. However, currently Tripal does not offer an efficient indexing and search solution for the contents stored in the Chado database, which largely limits the use of “big data”-oriented websites built on Tripal. Here we created two Drupal modules: the Elastic indexing module and the Elasticsearch module. These two modules take advantages of the powerful, full-text search engine Elasticsearch4, providing accurate and efficient indexing and searching for both Chado and Drupal database content. The Elastic indexing module allows website builders to select and index fields from any Chadoor Drupal database tables. The Elasticsearch module allows website users to build search interfaces for all Elastic-indexed tables of interest. With this module, users can implement advanced search methodology such as fuzzy matching, phrase matching, regular expression pattern searching, and range searching across fields from up to three joined tables. The beta versions of these modules have been developed within the Hardwood Genomics Database (hardwoodgenomics.org) and the code is available from GitHub (https://github.com/MingChen0919/DrupalElastic.git).

Reference: 1. Ficklin, Stephen P., et al. “Tripal: a construction toolkit for online genome databases.” Database 2011 (2011): bar044.

2. Drupal – Open Source CMS. https://www.drupal.org/ Accessed Feb 1, 2016.

3. Mungall, Christopher J., and David B. Emmert. “A Chado case study: an ontology-based modular schema for representing genome-associated biological information.” Bioinformatics 23.13 (2007): i337-i346.

4. Elastic. https://www.elastic.co/ Accessed Feb 1, 2016.

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7. Investigating the computational parameters used to model aquacobalamin in the CoII state using density functional theory Sarah J. Cooper1,2, Ryne C. Johnston1, Jing Zhou1,2, Jeremy C. Smith1,2, and Jerry M. Parks1,2 1 UT/ORNL Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory 2 Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, Tennessee Cobalamins, such as vitamin B12, act as cofactors employed by enzymes like isomerases and methyltransferases, giving them a vital role in energy production and metabolism. These molecules feature a cobalt atom bound to a corrin ring decorated with various amide groups and a 5,6-dimethylbenzimidazole (DMB) group that can covalently interact, occupying the lower axial position (β-ligand). The CoIII state exists as a hexacoordinate structure comprised of the equatorial tetracoordinate corrin ring and two axial groups, while in the crystal structure the Co II state exists as a pentacoordinate structure as a result of β-ligand dissociation. Previous work in modeling the CoII state has has been unable to show this pentacoordinate geometry. Here we use density functional theory to model and compare Co II and CoIII states of aquacobalamin using N-methylbenzimidazole to represent the DMB group. Several different parameters were tested in an attempt to form the pentacoordinate structure by introduction of explicit water molecules and explicit solvent, use of different basis sets, and the addition of dispersion corrections. The interaction energies and bond distances of the axial ligands were calculated to determine whether or not a pentacoordinate structure was achieved.

The results showed that the axial ligands in aquacob(III)alamin are strongly bound to the cobalt center and their bond lengths are rigid, irrespective to the computational parameters used. Although aquacob(II)alamin is formally pentacoordinate in crystal structures, we find that in biotic systems, water molecules loosely associate with and partially fill the vacant CoII β-site. The variability in the CoII β-ligand bond lengths underscores the necessity to thoroughly benchmark computational methods for predicting enzymatic mechanisms.

8. Synthetic bases alter the rate and quantity of methane produced by Methanosarcina barkeri Amanda DeVolk, Jun Yan, PhD., Frank Loeffler, PhD. Genome Science & Technology Methane is responsible for at least 10% of greenhouse gas emissions in the United States alone, and although not the most abundant, it is one of the most dangerously potent greenhouse gases. Methanogens are microbial organisms that utilize organic compounds, known as lower bases, to synthesize methane. It has been demonstrated that the rate and quantity of methane synthesis is dependent on the combination of lower base and methanogen. We hypothesize that exposing a methanogen to a non-ideal lower base, will alter the rate or quantity of methane it is able to produce. We will bombard the methanogen, Methanosarcina barkeri, with a variety of synthetic bases, and measure the rate at which the organism is able to produce methane. We hope to identify whether there is an increase or decrease in overall methane production, as well as a change in rate compared to the standard. The ability to speed up the rate of methane production could have considerable value in alternative energy research, while slowing down methane production may serve as a way to reduce the release amount of greenhouses gases produced.

10. Optimization of 2D- chromatographic conditions for deeper proteome coverage and higher duty cycle in MudPIT proteome experiments Ramsunder Iyer2; Richard J. Giannone1; Robert Hettich1 1 Oak Ridge National Laboratory, Oak Ridge, TN; 2University of Tennessee, Knoxville, TN Novel Aspect: Optimization of salt gradient in a MudPIT experiment for deeper proteome coverage with higher duty cycle measurements for microbial-community samples.

Introduction: A standard MudPIT approach involves a series of distinct salt pulses in which the concentration of ammonium acetate is increased linearly in successive steps. Each salt pulse displaces a sub-population of peptides from SCX to the RP, which is then analyzed by the mass spectrometer. We have developed a modified scheme in which initial pulses were significantly lower in concentration as compared to the conventional pulse scheme used routinely. This modified scheme enhanced the separation between adjacent salt pulses, leading to an improved distribution of peptides across the reverse phase resulting in their better detection. This enabled an extension of this modified scheme to improve the duty cycle of the MS measurement, without impacting the overall quality metrics.

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Methods: This approach was tested on a mixture of six environmental microbes (Saccharomyces cerevisiae, Escherichia coli, Clostridium thermocellum, Ignicoccus hospitalis, Nanoarchaeum equitans, and Streptomyces eurocidicus). This complex sample was chosen to test the robustness of our approach. Proteins were extracted by an SDS-based cellular lysis followed by TCA-precipitation. The samples were subjected to trypsin digestion and were loaded onto a 2D column consisting of SCX and RP. Following proteolysis, peptides were analyzed by 2D-LC-MS/MS on LTQ-Velos mass spectrometer (ThermoScientific). The resulting raw files were converted to mzML peaklists by ProteoWizard MSConvert and database search done using MyriMatch. IDPicker was used for protein assembly with a maximum FDR of 2% and minimum of 2 unique peptides to identify a given protein.

Preliminary Data: Our optimized MudPIT approach exploited seven shallow salt pulses (10-50 mM ammonium acetate), followed by five larger step pulses (75-500 mM). We found this to dramatically enhance the separation of the large plug of peptides eluting at the initial stage. To test the possibility of improved duty cycle proteomic measurements, we designed 15 hr, 13 hr and 9 hr modified LC schemes. Success was evaluated by comparing these shortened time schemes with the conventional scheme employing a total runtime of 22 hrs. These shortened times were chosen based on the desire to achieve at least two deep MudPIT experiments per day without impacting the overall proteomic metrics. A total of 16,710 distinct peptides, 3,201 distinct proteins and 102,797 spectra were obtained with the 15 hr modified MudPIT experiment (average of three technical replicates). For the 13 hr modified MudPIT scheme, the peptide, proteins and spectral counts were 16,159, 3164 and 70067 respectively. These numbers were strikingly similar to the conventional 22 hr MudPIT scheme (15599 distinct peptides, 3206 distinct proteins and 98074 spectra), indicating that time reduction by nine hours did not have deleterious effects on proteomic measurements. Preliminary analysis of 100 low abundance peptide species of the conventional 22 hr and modified 13 hr indicated that 42 peptides had a higher spectral count in the modified scheme as compared to the conventional scheme. Thus, a somewhat enhanced dynamic range is achieved in the modified scheme, allowing the low abundance peptides more chance for detection. There was no noticeable bias in protein detection for individual microbes with the 15 or 13 hr schemes. Thus, we found that either of the 15 hr or 13 hr modified MudPIT approaches were comparable with the much longer conventional approach, and thus yielded a significant increase in measurement duty cycle.

11. Characterization of an aminoglycoside modifying enzyme, aminoglycoside N3 acetyltransferase-Via Kumar Prashasti 1) and Engin Serpersu 1) 2) 1) Graduate School of Genome Science and Technology, The University of Tennessee and Oak Ridge National Laboratory, Knoxville, Tennessee 37996 2) Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996 Aminoglycoside (AG) antibiotics are bactericidal agents used to treat various bacterial diseases like tuberculosis, meningitis and hospital borne infections. However, the clinical effectiveness of these drugs has been drastically affected by the emergence of resistant bacterial strains, which is achieved by plasmid-encoded enzymes called aminoglycoside modifying enzymes (AGMEs). These enzymes can covalently modify their substrates, rendering them ineffective. AGMEs show variable levels of substrate promiscuity, but no correlation has been confirmatively observed between the sequence or structure of an AGME and its substrate profile. Our goal is to understand the molecular principles underlying this ligand selectivity by deciphering the thermodynamic and dynamic properties of enzyme-ligand complexes.

In this work, we describe kinetic, dynamic and thermodynamic properties of the aminoglycoside N3 acetyltransferaseVia (AAC-Via). AAC-Via catalyzes the modification of N3 atom of the 2-deoxystreptamine (2-DOS) ring on aminoglycosides by transferring an acetyl group from acetyl CoA. Despite significant sequence similarity to other aminoglycoside acetyltransferases, unlike others, it has a very limited substrate profile and therefore it presents an excellent opportunity to study molecular basis of ligand promiscuity.

Substrate profile of AAC-Via was observed to be limited to four substrates; gentamicin, sisomicin, tobramycin and kanamycin B. Analytical ultracentrifugation (AUC) studies demonstrated that the proportion of dimeric enzyme increases with increasing concentrations of enzyme. AUC studies also showed that dimer formation was mainly due to polar interactions which become weaker in the presence of salt. Binding of ligands favor the monomeric form of AAC-Via. Isothermal titration calorimetry (ITC) studies showed that ligand binding is enthalpically driven, entropically unfavorable and is accompanied by decreases in pKa’s of titratable groups. Studies in D 2O and H2O showed that solvent effect plays a role in the heat capacity exchange during the formation of binary complex, but no solvent effect is observed for ternary complex formation.

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AAC-Via thus behaves similar to some of the previously characterized more promiscuous AGMEs thermodynamically, and yet exhibits different kinetic and oligomerization properties. This study, in combination with exploring the structural and dynamic properties of AAC-Via, will provide a better understanding of the principles underlying ligand promiscuity.

12. Caldicellulosiruptor bescii DSM6725 stress responses to pH and osmolarity variation Punita Manga1,2, Kyle Sander2, Miguel Rodriguez Jr.2,3, Dawn M. Klingeman2,3, and Steven D. Brown1,2,3,§.

1 Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, Tennessee, USA; 2 BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA; 3 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA; § Corresponding author Caldicellulosiruptor bescii is a hyper thermophile (70-80oC) that can utilize wide range of substrates. However, inhibitors present in biomass, generated through pretreatment or as a product of metabolism can limit growth and bioconversion to products. Inhibitory chemicals such as sugar or lignin degradation products and weak acids are examples of compounds that are known to adversely affect C. bescii growth. Complete inhibition of C. bescii growth at 200mM concentrations of organic acids such as acetic acid (Baronofsky et.al., 1984) and 50mM NaCl (Farkas et.al.

2013) as well as fermentation ends around pH 5.0 have been previously reported. A systematic study into C. bescii inhibitors will provide a deeper understanding of physiology and regulation of the organism’s stress response and may generate targets for applied strain development goals. In this study, we examined responses to acid and osmotic stresses using various compounds to study C. bescii growth and physiology. C. bescii DSM6725 was grown in fermenters at 75 0C and at mid-log phase the pH was lowered to 6.0 for treated group using sulfuric acid while controls cultures were maintained at pH 7.2. Surprisingly, increased cell growth was observed in treated fermenters as compared to controls. RNA-seq data analysis using DESeq2, from samples collected pre and post acid stress detected ABC transporters, especially the xylose sugar ABC transporters, as the most significantly differentially expressed genes. To test dependence of increased growth in fermenters on sulfate from sulfuric acid, bottled experiments with and without sodium sulfate were conducted and negligible growth differences were observed. pH optima experiments are ongoing and will be reported. Preliminary NaCl osmolarity in the range 150-350 mOsm indicated longer lag phases above 200 mOsm with similar end-point growth densities across range and form the basis for more detailed ongoing studies.

15. The Effect of Charge Model Construction on Biomolecular Brownian Dynamics Simulation Results John Ossyra and Dr. Xiaolin Cheng Genome Science and Technology The specific charge distribution of biomolecules have been shown to accelerate and correctly orient partner binding surfaces by steering them together during diffusional association. Biomolecular Brownian Dynamics Simulation is used to study these binding processes by animating the intermolecular configurations through time, using simplified Effective Charge Models to provide the electrostatic interactions in the simulation. These models are an essential development for increasing the simulation speed to a practical level, however they are often left as a highly reduced charge description and may contain large calculation artifacts. As the simulation method matures to include more thorough treatments of biomolecular flexibility and interactions, one additional strategy is to extend the utility of this existing model. By considering additional parameters in constructing the charge models, the quality and consistency of the resulting binding structures can be improved.

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17. Optimizing an Extraction Method for Extending the Depth of Proteome Characterization of Microorganisms in Environmental Soil Samples Chen Qian1; K. Chourey2; R. Hettich2 1 University of Tennessee, Knoxville, TN, USA; 2Oak Ridge National Laboratory, Oak Ridge, TN, USA Microbial community proteomics is a powerful method to investigate microbial compositions and activities in natural ecosystems. There are several different protein extraction methods that have been used for soil proteomics characterization; however, limited microbial biomass in the presence of abundant interferences in soil samples still presents major challenges to mass spectrometry measurements. To obtain a more extensive and unbiased proteome characterization from microbes in soils, we have designed and optimized a revised extraction method to minimize interferences and enhance microbial protein detection. Our approach employed in-situ detergent-based microbial lysis and subsequent proteolytic digestion, but also added a processing step to clear the interfering humic acids by lowering the pH of the peptide sample to differentially precipitate the humic acids but leave the peptides in solution. The sample was then passed through a 10 Kda cutoff filter to retain the humic acids on the filter and thus remove them from the cleared peptide solution. We developed this approach using normal topsoil which is enriched in humic substances and inoculated with genome-sequenced bacteria (Pseudomonas putida F1, Pseudomonas putida KT2440, Shewanella oneidensis MR1 and Shewanella putrefaciens CN-32). Samples containing humic acids are typically brown colored and interference with colorometric protein quantification. After applying our method, the sample is clear and amenable to peptide quantification. Mass spectrometry identified around 1300 proteins for the single isolate Pseudomonas putida F1 and around 3500 proteins for four isolates mixture, both containing abundant humic acids in the sample.

These numbers are almost the same as control sample without humic acids, which means our humic acids removal step did not impact the protein identification. Notably, comparison of control and processed samples also confirmed that the humic acid removal step did not bias peptide detection. We have extended this in-situ proteome extraction approach to compare to an indirect protein extraction method (differential centrifugation). This has permitted us to evaluate and contrast planktonic vs. sediment-attached microbial populations in these real-world samples.

Key words: soil microbial proteomics, protein extraction method, humic acids removal

18. Aqueous Yerba Mate Extract Alters Cell Membrane Integrity and Catalase Activity of Salmonella Typhimurium DT104 Strain 2576 Caroline S. Rempe1, Kellie P. Burris2, Scott C. Lenaghan3,4, and C. Neal Stewart, Jr.2 1 Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996, USA 2 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 3 Center for Renewable Carbon, University of Tennessee, Knoxville, TN 37996, USA 4 Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, USA Email: crempe@vols.utk.edu Salmonella Typhimurium is a gram-negative enteropathogen that infects millions worldwide and the rapid emergence of antibiotic resistant strains has heightened the urgency of developing effective treatment for this pathogen. Previous studies have demonstrated that the aqueous extract of yerba mate (Ilex paraguariensis) provides effective therapy, in vitro, for the drug-resistant S. Typhimurium DT104. Unfortunately, while some of the chemical constituents that contribute to the extract’s antibacterial activity have been identified, the mechanism of action of the extract is still unknown and of crucial importance to its future potential in combating antibiotic resistant bacteria. In this work, it was hypothesized that membrane leakage and increased reactive oxygen species (ROS) were the primary mechanisms that contributed to the antibacterial activity of the yerba mate extract. As such, the integrity of the cell membrane, after exposure to yerba mate extract, was quantified using a crystal violet assay, while the presence of ROS was indirectly measured as a function of catalase activity. With a ca. 8-9 log colony forming units/ml starting bacterial population, the results indicated that cell membrane integrity was compromised within the first hour of growth while catalase activity was significantly reduced after 4 h with an inhibitory dose (80 mg/ml) of yerba mate extract. This data suggests that membrane leakage, but not ROS, may be responsible for the antibacterial activity of yerba mate extract. Due to the strong antioxidant properties of the yerba mate extract, it is possible that the extract itself prevents accumulation of ROS, since extract alone did not inhibit the activity of a catalase control. Further studies into the expression of key genes involved in response to ROS may shed further light on the impact of ROS as a mechanism of action for yerba mate extract.

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19. Novel derivative of doxorubicin, AD198 for treatment of bladder transitional cell carcinomas in vitro.

Dmitriy Smolensky1,2, Kusum Rathore1, and Maria Cekanova1,2* 1 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee, 37996, USA 2 UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, Tennessee, 37996, USA * Corresponding Author: mcekanov@utk.edu Human bladder cancer is one of the most expensive cancers to treat due to its high rate of reoccurrence. Development and characterization of animal models for human cancers is important for the improvement of diagnosis and therapy.

The bladder transitional cell carcinoma (TCC) of domestic animals resembles human bladder TCC in many aspects, therefore cell lines derived from bladder TCC of dogs are valuable model for studying human bladder TCC. A chemotherapeutic agent, Doxorubicin (Dox), is often used to treat advanced bladder cancer. Despite the success of Dox based therapies, drug resistance and cardio-toxicity are limiting factors in treating any cancer with Dox. Nbenzyladriamycin-14-valerate (AD198), a novel derivative of Dox has no measurable cardio-toxic effects in the rodent model of lymphoma. In this study, we focused in comparing the efficacy and mechanisms of Dox and AD198 in human and primary canine bladder transitional cell carcinoma (TCC) cells in vitro. Using human T24 and UMUC-3 TCC and three canine primary bladder K9TCC-Dakota, K9TCC-Lillie, and K9TCC-Molly cell lines, we evaluated the Dox and AD198 efficacy on cell proliferation by MTS assay. Caspase 3/7 assay, reactive oxygen species (ROS) production, and western blot analysis were used to study the mechanisms of Dox- and AD198-induced apoptosis.

AD198 was more effective than Dox in inhibition of cell proliferation in tested TCC cells in dose-dependent manner.

ROS production was increased when compared to control by both Dox and AD198 leading to apoptosis, which was confirmed by caspase 3/7 activity and cleavage of poly ADP ribose polymerase (PARP) in tested TCC cells. AD198 increased ROS production significantly more than Dox in all tested TCC cells. Both Dox and AD198 increased phosphorylation of pro-apoptotic p38 MAPK, while at the same time increased the activity of anti-apoptotic phosphatidylinositol 3-kinase (PI3K/AKT/mTOR) signal transduction pathway in human TCC cell lines. When a PI3K inhibitor, LY294002, was used in combination with either Dox or AD198, cell proliferation was inhibited more effectively than when drugs used alone. In addition, cleaved PAPR was increased when TCC cells were co-treated with LY294002 and in combination with Dox or AD198 as compared to either drug alone. Our data indicates that AD198 as novel derivative of Dox may be a valuable treatment option for bladder TCC cancers. Dox- and AD198induced AKT phosphorylation that is an indicator of pro-survival and drug-resistance mechanisms of chemotherapies in tested bladder TCC cancer. Combined therapy of Dox or AD198 with inhibitors of PI3K/AKT1 pathway might lead to more effective treatment outcome for human and veterinary patients diagnosed with bladder TCC cancer.

Evaluation of new therapeutic and imaging drugs that have shown promise in vitro and in vivo in the rodent model are important; however, pet animals like, dogs and cats with naturally-occurring tumors provide an important step for successful translation from rodents to human clinical application.

20. Pathogenic conversion of Foxp3+ T cells into Th1 and Th17 cells in Stromal Keratitis Siva Karthik Varanasi1, Siddheshvar Bhela2 and Barry Rouse1,2 1 Department of Genome Science and Technology, University of Tennessee, Knoxville, TN, 37996-0845 2 Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996-0845 Ocular infection with herpes simplex virus 1 (HSV-1) can result in a chronic 13thane-inflammatory lesion that is a significant cause of human blindness. IFN-Îł producing CD4 + T cells are generally considered the main orchestrators, and lesions are more severe if the Foxp3+ regulatory CD4 T cell (Treg) response is compromised. Tregs have been shown to lose FoxP3 (lineage factor) and adopt alternate lineage fates in a changing cytokine environment. Moreover, little is known about the stability of Treg cells in an ongoing inflammatory reaction such as is the case of SK. In this study-using fate mapping mice, we were able to demonstrate that the population of ex-Tregs increased at the site of infection that is the cornea and also at the secondary lymphoid organs after HSV-1 infection. In vivo studies showed that these ex-Tregs acquired the Th1 and Th17 phenotype, which may play a destructive role in stromal keratitis (SK).

We also demonstrate in vivo that CD25lo subset of Tregs is less suppressive and more prone to conversion than CD25hi Tregs. Additionally, in-vitro studies suggested that Tregs in the presence of IL-12 converted into ex-Tregs, however this plasticity was prevented when Tregs were generated in the presence of DNMT inhibitor (Azacytidine).

Thus, plasticity of Tregs might represent a problem during SK, which needs to be controlled with appropriate therapeutic procedures. Overall, our results indicate that Tregs lose FoxP3 expression in ocular inflammatory setting and drugs that reverse Treg plasticity could be useful to restore Treg function and to inhibit ocular immunopathology.

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22. Selection of protein biomarkers in Dehaloccoides mccartyi strains enables an MRM-MS approach for monitoring dechlorination activities in environmental samples Manuel I. Villalobos Solis2; Karuna Chourey1; Frank Loeffler2, Robert Hettich1 1 Oak Ridge National Laboratory, Oak Ridge, TN; 2University of Tennessee, Knoxville, TN (23) Problem under investigation Different strains of the bacterium Dehaloccocides mccartyi (DhC) are able to metabolically transform toxic perchloroethene (PCE) into benign 14thane by means of organohalide respiration. This biological process presents an interesting opportunity for natural bioremediation strategies at sites contaminated with chlorinated compounds.

However, an appropriate qualitative and quantitative detection of the reductive dehalogenase enzymes (Rdases) expressed by these microorganisms is required for a better understanding of their action on different substrates. By using a combination of experimental and in-silico prediction data of the most likely detected peptides in enrichment cultures of DhC strains, we have selected bioindicators of these enzymes that could be monitored in environmental samples via multiple reaction monitoring-mass spectrometry (MRM-MS).

(2) Methods Tryptic peptide solutions of an enrichment BDI (strains CBDB1, GT, VS, BAV1, 195) and strain BAV1 microbial cultures amended with PCE and cis-dichloroethene (cis-DCE) respectively, were analyzed on an Eksingent NanoLC2D system coupled on-line with a TSQ Quantum Discovery MAXTM (ThermoScientific) triple quadrupole mass spectrometer equipped with a nano-electrospray source. Amounts ranging from 125 ng to 10 µg of digested material were loaded on a capillary back-column (12 cm x 150 µm, length x id) and separated on a capillary front-column (16 cm x 100 µm, length x id) packed with ~ 5 cm and ~ 16 cm of C18 Kinetex individually. Assessment and refinement of the targeted proteomics assay is conducted in Skyline v. 3.1 (http://skyline. Maccosslab.org).

(3) Preliminary results Targeted proteomics analyses of the PCE-to-ethene-converting BDI microbial consortium, using a combination of global proteomics data and predicted observability software, revealed a total of 71 doubly charged precursor and yfragment ions from ten proteins including those of three Rdases (VcrA, TceA, putative reductive dehalogenase) and other reported molecular bioindicators for general Dehaloccoides activity, such as chaperonin GroEL and formate dehydrogenase, alpha sub-unit (FdhA). Selected transitions were subsequently monitored in technical triplicates under different amounts of digested peptides, total areas under the curve plotted against loaded amount of peptides and CV values calculated. Linearity was observed between 500 ng and 8 µg of total digested sample for the majority of proteins tested. Additional experimentation on the same BDI culture but with an exclusive focus on Rdases, resulted in new peptide candidates and transitions found for these enzymes that had been excluded in the initial approach. Targeted analysis of DhC BAV1 pure cultures amended with cis-1-2-dichloroethene was also conducted, and this provided more information for peptide, precursor and fragment ion selection for biomarker proteins, including vinyl chloride reductive dehalogenase (BvcA) which was not studied before. Transitions that exhibited reproducibility in peak shapes and higher contributions to total AUCs in the enrichment cultures have now been extended in the targeted analysis of environmental groundwater samples known to harbor DhC cells. By using GroEL to monitor DhC presence, we were also able to detect trichloroethene reductive dehalogenase (TceA) in a groundwater sample obtained from an actively dechlorinating site in Brazil; however, further work is needed to optimize the range and stability of the MRM targets employed.

(4) Conclusion Integration of global profiling and in-silico prediction data of the most likely observable peptides in BDI and BAV1 enrichment cultures by MRM-MS provided targeted information of relevant proteins that is useful for their monitoring in environmental samples; however, preliminary work has demonstrated that peptide and transition target selection is critical, as the microbial taxa and protein complexity vary with respect to the enrichment cultures making their detection by MRM-MS more challenging due to increased charge suppression effects and numerous co-eluting peptides sharing similar transitions.

23. Gene, Sex and Diet Interact to Control the Tissue Metabolome Ann Wells1, Bill Barrington2, Stephen Dearth3, David Threadgill2, Shawn Campagna3, Brynn Voy1, 4 1 University of Tennessee-Knoxville, Genome Science and Technology, 2Texas A & M University, 3University of Tennessee-Knoxville, Department of Chemistry, 4University of Tennessee-Knoxville, Department of Animal Science Individuals use diet to lose weight, combat disease and improve overall health, but one diet does not necessarily fit all. Genetic variation is an important determinant of dietary efficacy, but little is known about how diet and genetics interact at the tissue level to alter metabolism. The objective of this study is to determine how dietary regimens that are known to impact health impact tissue level metabolism, and how these responses are influenced by genetic

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background, using untargeted metabolomics. Ultimately, metabolite profiles will be associated with overlying system traits linked to obesity and its consequences. Four strains of mice (C57Bl/6J, FVB/NJ, A/J and NOD/LtJ), which included males and females, were fed one of five isocaloric diets (Research Diets Inc): Mediterranean (D12052702), ketogenic (D12052706), Japanese (D12052703), Western (D12052705) or standard chow (D12052701). Abdominal adipose, liver and muscle tissue metabolites were measured using liquid-chromatography mass spectrometry.

Statistically significant differences between groups were identified using the following model: , where tissue weight and internal standard are covariates. ANOVA (FDR p-value<.05) showed that 116, 124, and 158 metabolites in adipose, liver and skeletal muscle, respectively, were significantly altered by sex, strain, diet, strain by diet, strain by sex, diet by sex, or sex by strain by diet interaction. Clustering algorithms were used to group mice based on â&#x20AC;&#x153;metabotypesâ&#x20AC;?, phenotypes defined by distinct strain and diet dependent metabolite profiles. Unsupervised clustering revealed that metabolite profiles distinguish mice based on strain. Supervised partial least squares-discriminant analysis showed that the effects of diet, in adipose, muscle, and liver tissue was most apparent with the ketogenic and western diet differentiating from the other four diets. The effects of sex and diet on all three tissues distinguished male and female Japanese diet from all other metabotypes. Calculating the VIP scores for all potential interactions revealed that the importance of certain metabolites were unique to each interaction. Diet by sex and diet in adipose and muscle tissue showed hydroxyproline, an amino acid found primarily in collagen, to be the most influential metabolite on clustering. Standard and ketogenic diets, regardless of sex, have lower peak abundances of hydroxyproline than all other diets. Allantoate and N-acetyl-L-ornithine were most influential to liver tissue. Japanese males have lower peak abundances of allantoate, while Japanese male and females have higher peak abundances of N-acetyl-L-ornithine than all other diets. Collectively, these data illustrate that macronutrient composition and genetic background interact to alter tissue metabolism independent of caloric intake. Ongoing efforts are directed toward linking metabotypes to systems level traits that are relevant to obesity and metabolism.

Biochemistry & Cellular and Molecular Biology Student Abstracts 24. Inhibiting matrix metalloproteinases 2 and 9 alters circadian neuronal firing patterns in the suprachiasmatic nucleus Abrahamsson KE1, Prosser RA1 Photic entrainment of mammalian circadian rhythms is a plastic process that occurs within the suprachiasmatic nucleus (SCN), or master clock. During the subjective night, exposure to light or in vitro application of glutamate shifts clock phase, which is seen in vitro as a shift in peak neuronal activity. The inability to synchronize internal clocks to the 24hour light cycle can result in jet-lag, shift work disorder, or increased risk of diabetes or cancer. Complex intracellular processes govern entrainment, but a variety of extracellular proteins can regulate photic/glutamate phase-shifts in the SCN. Matrix metalloproteinases, MMP2 and MMP9, generally serve as extracellular matrix remodelers and also participate in synaptic plasticity mechanisms. Because the role of MMP2/9 in SCN entrainment mechanisms is unknown, we sought to investigate MMP2/9 activity in SCN acute slices using in vitro, extracellular neuronal activity recordings. We hypothesized that blocking MMP2/9 would block glutamate-induced phase delays. Contrary to our initial hypothesis, in vitro application of the MMP2/MMP9 inhibitor, BiPS, alone can induce phase shifts, similar to the effects of glutamate. Additionally, phase shifts induced by BiPS were blocked by co-application of an NMDAR antagonist. Preliminary data showed that BiPS induced phase shifts after early-night and day-time application, but not after late-night treatments. Currently we are investigating how MMP2/MMP9 modulates NMDAR signaling in the SCN and if MMP2/9 are necessary for the maintenance of circadian rhythms. Combining the aforementioned studies with temporal assessments of MMP2/MMP9 expression and activity in both SCN tissue and in the SCN2.2 cell culture line should clarify the role(s) of these proteases in the SCN. 25. The role of ethylene response sensor protein slr1214 during Phototaxis in Synechocystis sp. PCC6803 Cidney Allen, Randy Lacey, and Brad Binder The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 moves in response to light in a process known as phototaxis. This response causes the cells to move toward (positive) or away from (negative) a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how these photoreceptors affect single-cell motility behavior. Previous research in the Binder lab identified an ethylene receptor, SynEtr1, that affects phototaxis and appears to signal via slr1214. It was also previously observed that the age of cells affect motility and the effect of ethylene on motility. Therefore, we examined slr1214 in younger and older cells to test the hypothesis that slr1214 affects motility. We also examined slr1214 in two strains, one from a lab in Japan and

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another from the Pasteur Institute in France. To do this, we used qPCR to determine the expression of slr1214 of cells phototaxing in air versus ethylene. The results from these experiments will provide an indication on how ethylene is affecting slr1214 in both strains of WT Synechocystis and how this relates to motility.

26. The Protease Activity of Separase Is Required for Both Chromosome Segregation and Regulation of Membrane Trafficking During Mitotic and Meiotic Exocytosis Xiaofei Bai, Diana Mitchell, Lindsey Klebanow, Joshua N. Bembenek Dr. Bembenek Lab from Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville Chromosomal segregation and cytokinesis are tightly regulated processes involved in cell division. A crucial protein involved in this process is a cysteine protease separase which is required for proper chromosomal segregation. This separase cleaves a protein complex called cohesin, which keeps the sister chromatids together until the end of metaphase. Currently, separase is known to be a key player in meiotic cortical granule exocytosis and mitotic cytokinesis process. Additionally, in previous studies we have shown that RNAi knockdown of separase impaired RAB-11 vesicular trafficking. RAB-11 is a small GTPase that also regulates both cortical granule exocytosis and exocytosis during cytokinesis. This raises the question of whether or not separase’s protease activity regulates exocytosis via a RAB-11 dependent pathway. To further understand separase’s regulatory role in chromosome segregation and exocytosis, we investigated a mutant with point mutation in the catalytic activity site named SEP1PD::GFP. We investigated this protease-dead separase mutant utilizing a GFP tagged variant with a spinning disk confocal microscope to observe cellular phenotypes. We found that the expression of SEP-1PD::GFP causes chromosomal nondisjunction. Depletion of cohesin rescues this defect, indicating that cohesin cleavage is prevented by the inactive protease, possibly by a substrate trapping mechanism. To test whether SEP-1PD::GFP also impairs RAB-11 vesicular trafficking during cytokinesis, we imaged the embryos that express RAB-11::mCherry and SEP1PD::GFP. Interestingly, expression of SEP-1PD::GFP causes an abnormal accumulation of RAB-11 vesicles at the cleavage furrow site, similar to depletion of separase by RNAi. Moreover, we found that RAB-11 co-localized with both wild type separase and our protease dead mutant on cortical granules during meiotic anaphase I. To further examine whether or not protease activity is required for exocytosis, we filmed SEP-1PD::GFP expressing embryos during meiosis I to observe cortical granule exocytosis. Our results indicated that SEP-1PD::GFP expression delayed the completion of cortical granule exocytosis. To obtain further insight into the molecular mechanism of protease activity of separase during exocytosis, we tested possible genetic interactions between SEP-1PD::GFP and core exocytic proteins. Our results indicate that the depletion of the syntaxin-4 (syx-4), a plasma membrane localized tSNARE required for exocytosis, further enhanced RAB-11::mCherry and SEP-1PD::GFP accumulation on the cleavage furrow and the midbody. Moreover, we found a synergistic increase in embryonic cytokinesis failure relative to syx-4 depletion or SEP-1PD::GFP expression alone. These findings suggest that the protease activity of separase is required for the exocytosis of RAB-11 vesicles during mitotic cytokinesis and cortical granule exocytosis during meiosis I.

27. Quantification and Interpretation of Weak Preferential Interactions between Folate and Betaine Purva P. Bhojane, Michael R. Duff, Jr., Gabriella Rimmer, and Elizabeth E. Howell Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville In vitro studies with two different dihydrofolate reductases (EcDHFR, E.coli chromosomal and R67 DHFR, plasmid encoded) have shown that weak interactions between osmolytes and the substrate, dihydrofolate (DHF), decreases the affinity of DHF towards these enzymes. The unique changes in binding affinity with water activity for each osmolyte indicate preferential interactions between osmolyte and folate and its derivatives. Characterization of these interactions is essential for better understanding of in vivo effects of folate and its various redox states with available functional groups inside the cell. Quantitation of weak interactions using a vapor pressure osmometry method yields a preferential interaction coefficient, or μ23/RT value. This provides a scale for measuring the preference of folate for betaine relative to water. Experimental measurements found a folate concentration dependence of the μ 23/RT values, consistent with dimerization of folate. Our results also indicate neutral folate preferentially interacts with betaine whereas the anionic form excludes betaine. Studies with other model compounds suggest aromatic rings prefer to interact with betaine as compared to water. The preferential interaction coefficients or μ 23/RT values obtained for additional nitrogen containing aromatic compounds were dissected into additive contributions from chemically distinct functional groups.

The atomistic interaction potentials for each of the surface types (alpha values) were calculated. The calculated set of values coupled with the water-accessible surface areas (ASA) can be used to predict the μ23/RT of any compound with

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betaine. Additionally, solubility assays were done to quantify the free energy of transfer of folate from water to 1 M betaine solution. Data indicate favorable interactions between betaine and folate at a lower pH with a negative free energy of transfer whereas at higher pH, the free energy of transfer is positive. These results are consistent with our µ23/RT dependence of pH. Can μ23/RT values be used to predict osmotic stress effects on ligand binding? In some cases, yes. However, the caveat is whether all the ligand atoms are used in binding. As glutamate excludes betaine, calculation of the μ 23/RT value for polyglutamylated folates (pteroyltetra-γ-glutamate (PG4)) predicts an overall exclusion of betaine. This should translate into tighter binding of PG4 to DHFR. Our studies found betaine addition weakens binding of both folate and PG4 to R67 DHFR to similar extents. This result indicates the polyglutamylated tail does not contact the enzyme.

29. tPA, uPA, and LRP-1 regulate glutamate-induced phase resetting of the mammalian circadian clock.

Cooper JM. & Prosser RA Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, USA The mammalian circadian clock in the suprachiasmatic nucleus (SCN) exhibits endogenous rhythms in neuronal activity and synaptic plasticity, regulates daily rhythms, and entrains to external stimuli. Entrainment to light involves glutamate release from retinal ganglion cells onto SCN neurons, which activates NMDA receptors and initiates signaling to modulate clock genes. Intriguingly, responses to glutamate vary across the circadian cycle: glutamate at night induces an advance or delay in clock phase, while daytime glutamate has no effect. The mechanisms underlying this duality are not fully understood. The plasminogen activating system influences neuronal responsivity throughout the brain, and our research shows this system regulates glutamate signaling in the SCN. Tissue-type plasminogen activator (tPA) activates plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. We are studying additional proteins within the plasminogen system, investigating 1) whether urokinase plasminogen activator (uPA) acts as a redundant mechanism within the SCN; and 2) whether low density lipoprotein receptor-related protein 1 (LRP-1) regulates phase shifting via interactions with tPA. SCN brain slices from adult male C57BL/6 or tPA knockout (tPA-/-; B6.129S2-Plattm1Mlg/J) mice were treated with glutamate +/- LRP-1 inhibitors (RAP or anti-LRP-1 antibody) at ZT16 or ZT23 (ZT0=lights-on). The following day we recorded SCN neuronal activity to determine clock phase. Control SCN slices from adult tPA-/- mice exhibit entrained neuronal activity rhythms, and glutamate at ZT16 and ZT23 induces phase delays and advances, respectively. Thus, tPA -/- mice do not exhibit severe deficiencies in clock phase regulation, possibly reflecting redundant mBDNF-generating pathways. The tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), prevents phase shifting in the tPA-/mice, and casein-plasminogen zymography reveals a day-night rhythm in uPA expression. These data suggest uPA compensates for the absence of tPA in tPA-/- mice. Addressing the actions of LRP-1 in the SCN, concurrent application of RAP or anti-LRP-1 with glutamate to WT SCN slices prevents normal shifts, while RAP alone has no effect. RAP continues to inhibit glutamate phase resetting in tPA-/- slices, indicating LRP-1 may influence the clock independently of tPA. These results elucidate SCN phase shifting mechanisms and more broadly address plasminogen activator regulation of neuronal responses.

31. Characterizing the role of RPS6 phosphorylation in Arabidopsis thaliana Ramya Enganti1 and Albrecht von Arnim1, 2 1 Biochemistry, Cellular and Molecular Biology and 2Genome Science and Technology The University of Tennessee Knoxville, TN 37996, USA Translational regulation in eukaryotic cells is a key process required to maintain protein levels under various environmental conditions to regulate growth and development. One such protein subjected to translational control is ribosomal protein of small subunit 6 (RPS6), which as the name suggests, is part of the eukaryotic 40S subunit. It is highly conserved among all eukaryotes and has been shown to undergo phosphorylation at multiple sites in its Cterminal tail. The phosphorylation is regulated in response to various environmental stresses such as mitogens, cold, heat shock and hypoxia and also light conditions (in plants). While this has been observed in several eukaryotes, its functional significance is still unclear. Recent studies in mammals have provided evidence for RPS6 phosphorylation in playing a role in regulating cell size, translation and ribosome biogenesis, however, very little is known about its role in plants. Arabidopsis RPS6 is encoded by two genes, RPS6A and RPS6B, and the two proteins are functionally equivalent. Double knockout leads to embryo lethality, however, single rps6 mutants are viable but show developmental defects including pointed leaves, short primary roots and reduced pollen viability. We hypothesize that RPS6 phosphorylation plays a role in regulating cell growth and translation in Arabidopsis. To understand the role of this phosphorylation, we have designed several phospho-null (serine to alanine mutations) and phospho-mimic (serine

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to aspartate) constructs with various combinations of the mutated residues. We have also generated transgenic lines with the various versions of the mutated RPS6 in the mutant background. These transgenic lines are currently being used to carry out phenotypic analyses under various stress conditions.

32. Identifying the role of glucosinolates in intercellular trafficking via plasmodesmata in thioglucoside glucohydrolase (tgg) mutants.

Jessica Fernandez and Tessa Burch-Smith Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville Cellulosic cell walls are a major hurdle in plant intercellular communication. To overcome this challenge, plants have evolved channels called plasmodesmata to allow plant cells to communicate directly via the cytoplasm.

Plasmodesmata transport nutrients and signaling molecules including hormones, small RNAs, and proteins. Viruses have evolved to take advantage of the plasmodesmata to spread during systemic infection. In the Arabidopsis thaliana increased size exclusion limit 2 (ise2) mutant, there is increased intercellular trafficking. An increase in number of branched plasmodesmata have also been observed in ISE2 silenced tissues. THIOGLUCOSIDE GLUCOHYDROLASE 1 (TGG1) encodes a myrosinase and is responsible for catalyzing the hydrolysis of glucosinolates. Expression of TGG1 was significantly down regulated in ise2 embryos. This study analyzes the effects of TGG1 and homolog TGG2 on intercellular trafficking and analyzes the expression of myrosinase in ise2 mutants. Confocal microscopy was used to monitor GFP movement from cell to cell as a measure of intercellular trafficking. Expression of TGG1 and TGG2 was quantified in ise2 Arabidopsis thaliana mutants. The results from this analysis help us define the role of myrosinases in intercellular trafficking and shed insight on the role of ISE2 with myrosinase.

33. In vivo effects of macromolecular crowding on the binding affinity of dihydrofolate to dihydrofolate reductase.

Gabriel Fuente, Michael Duff and Elizabeth Howell Department of Biochemistry & Cellular and Molecular Biology University of Tennessee Knoxville Escherichia coli is capable of growing in low and high osmolality environments, causing a rapid change in the movement of water across its cell membrane. To deal with the loss of water, many osmotically stressed cells either uptake or synthesize small molecules, called osmoprotectants. Examples of osmoprotectants include proline, betaine, trehalose, and glutamate. As a result, the water activity in the cells is decreased, leading to higher macromolecular crowding. We are interested in understanding how folate metabolism enzymes work under crowded conditions.

Dihydrofolate reductase is an enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). DHFR is an important enzyme in folate metabolism as generation of THF is required for the synthesis of thymidylate, methionine and other metabolic intermediates. Two types of DHFR exist, the chromosomally encoded DHFR and the plasmid encoded R67 DHFR. Previous in vitro osmotic stress studies showed that upon addition of various osmolytes a tighter binding of the NADPH cofactor and weaker binding of DHF was observed in both enzymes. However, in vitro studies are far from representative of the heterogeneous and crowded intracellular environment. A crucial difference between in vivo and in vitro conditions is the high concentration of macromolecules, which can range in cytoplasm from 200 mg/mL in eukaryotes to >400 mg/mL in prokaryotes. In contrast, most biochemical studies are conducted under dilute (<10 mg/mL) macromolecular conditions. These arguments lead to the longstanding question as to what extent do the experiments observed in vitro reflect the behavior in vivo6? In this work we will address this question by determining the binding affinity of DHF to DHFR in the interior of E. coli cell

by NMR. Here, an osmotic stress approach will be used to determine the in vivo effects of the ligand binding on the 19 F-labeled tryptophans of chromosomal DHFR and R67 DHFR. NMR will used to monitor the K d for the DHF binding to DHFR as a function of folate concentration. Thus far, we have standardized the purification of R67 DHFR and we are in the process of labeling our protein.

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34. Reviving antibiotics: Efflux pump inhibitors with a novel mechanism of action Adam T. Green1,2, Jerry M. Parks2, Khushboo Bafna3, Jerome Baudry1,2, Helen Zgurskaya4, and Jeremy C. Smith1,2 1 Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville 2 University of Tennessee/Oak Ridge National Laboratory Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory 3 Genome Science and Technology Program, University of Tennessee, Knoxville 4 Department of Chemistry and Biochemistry, University of Oklahoma, Norman The resistance of bacteria to many antibiotics is a growing public health concern. One of the most prominent mechanisms in Gram-negative bacteria involves efflux pumps that expel various antibiotics from the cell. In Escherichia coli, the AcrABâ&#x20AC;&#x201C;TolC efflux pump is responsible for resistance to fluoroquinolone antibiotics. AcrA facilitates the assembly of the functional tripartite complex by interacting with the other two components. The X-ray crystal structure of AcrA lacks its membrane-proximal (MP) domain, which has been shown to play a role in the assembly of the pump. Thus, we generated homology models of full-length AcrA and performed molecular dynamics simulations on each model. From the simulations and previous work, we identified a hinge-bending motion in AcrA. We hypothesized that this hinge region could serve as a binding site for small molecules that could disrupt the pump, prevent efflux, and restore the activity of existing antibiotics. To address this hypothesis, we identified 27 representative structures from the MD simulation trajectories and performed virtual screening of three libraries of small, drug-like molecules to two potential binding sites on AcrA. These sites included the hinge region and a hydrophobic pocket in the MP domain. We ranked each compound and the top hits were assayed experimentally.

From the virtual screening results, experimental assays confirmed 11 compounds that potentiate the activity of known antibiotics in vivo. Of these 11 compounds, five were confirmed to bind to AcrA by surface plasmon resonance. Three other compounds were also confirmed to bind to AcrA, but did not restore in vivo antibiotic activity. Ultimately, the identification of new binding sites has led to the discovery of small molecules that have the potential to overcome antibiotic resistance through a previously unrecognized mechanism.

36. Distinct domains confer CheA with unique functions in chemotaxis and cell length at division in Azospirillum brasilense Sp7 Jessica M. Gullett, Amber Bible, and Gladys Alexandre Department of Biochemistry, Cellular, and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase, CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium, Azospirillum brasilense, the Che1 operon controls swimming speed during chemotaxis as well as changes in cell size at division. However, the exact molecular mechanisms that allow this chemotaxis system to modulate multiple cellular behaviors are not known. Here, we identify specific domains of CheA1, the histidine kinase from the Che1 operon, that contribute to each of these functions. We show that CheA1 is produced in two forms: a fusion protein with a conserved seven-transmembrane domain of unknown function at the N-terminus, and a truncated, soluble CheA1. Using fluorescence microscopy and subcellular fractionation, we show that soluble CheA1 localizes at the cell poles while membrane-bound CheA1 is distributed throughout the cell surface. Site-directed and deletion mutagenesis combined with behavioral analyses show that the conserved histidine residue of CheA1 exclusively regulates chemotaxis responses while the conserved N-terminal transmembrane domain is necessary and sufficient to modulate changes in cell size.

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37. The Cdc42 GEF Scd1 regulates septum formation during cytokinesis in S. pombe Brian Hercyk, Bin Wei, and Maitreyi Das Department of Biochemistry, Cellular, and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 Cytokinesis is a fundamental process that must be properly executed in all organisms to maintain genome integrity; defects in this process contribute to developmental disorders and may lead to the onset of cancer. Following chromosome segregation, cytokinesis occurs in a sequential process that involves contractile ring assembly, constriction, furrow ingression, and cell abscission. In yeast, furrow ingression is concurrent with septum (cell wall) formation. It is not clear how the actomyosin ring and septum formation occur concurrently. Our results demonstrate that the Cdc42 GEF’s (Guanine nucleotide Exchange Factor), Gef1 and Scd1, sequentially activate Cdc42 at the site of cell division in a unique spatiotemporal manner and promote distinct cytokinetic events. We investigated the role of the GEFs during cytokinesis using classical genetics and fluorescent microscopy. We find that the GEF Gef1 at the actomyosin ring is required to initiate timely onset of ring constriction and septum ingression. In contrast, Scd1localizes to the ingressing membrane during cytokinesis, where the septum is formed. We find that scd1Δ mutants display aberrant septa, with thicker septa that tend to bend and buckle. Further, electron micrographs of scd1Δ cells fail to show a distinct primary septum. Consistent with this observation, at the septa of scd1Δ cells we find decreased levels of the primary septum building enzyme Bgs1, while there is a modest increase of the secondary septum building enzyme Bgs4. Furthermore, scd1Δ cells expressing fluorescently tagged Bgs1 contain multiple septa, indicating cell separation defects. Together these results indicate that Scd1 plays a role in promoting primary septum formation and restricts secondary septum formation. Thus Scd1 likely coordinates different events during septation in cytokinesis.

Further studies will indicate the molecular details of Sdc1 function during cytokinesis.

38. The role of solvent effect on the aminoglycoside-binding properties of thermophilic and mesophilic variants of Aminoglycoside Nucleotidyltransferase 4' (ANT4) Seda Kocaman, Xiaomin Jing and Engin H. Serpersu Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996.

Aminoglycosides are a group of antibiotics that bind to the 16s ribosome RNA of prokaryotes and thereby cause mistranslations and premature stops which interfere with protein translation. Some bacterial species developed resistance against aminoglycosides due to their Aminoglycoside modifying enzymes (AGMEs). Aminoglycoside nucleotidyltransferase 4' (ANT4) is an AGME which transfers the AMP group from MgATP to the C4’-OH of aminoglycosides in order to detoxify them. Two thermostable variants (T130K and D80Y) of ANT4 have been created in our laboratory by introducing single point mutations to the wild type (WT) ANT4. Our group has shown earlier that; local flexibility and ligand binding properties of T130K resembles the mesophilic WT enzyme whereas D80Y which has the highest melting temperature presents distinct features. This work is based on the hypothesis that solvent reorganization upon ligand binding may be used to identify properties that lead to thermophilicity. Since the active sites of ANT4' is highly solvent exposed, binding of ligands will cause (or affected by) differential solvent reorganization. Therefore, we are investigating the solvent reorganization on the aminoglycoside-binding properties of different ANT4 variants via performing isothermal titration calorimetry (ITC) both in light water (H2O) and heavy water (D2O).

39. Ethylene Stimulates Phototaxis in Synechocystis sp. PCC6803 Through the Ethylene Receptor SynETR1 Randy Lacey and Brad Binder BCMB Department, University of Tennessee-Knoxville Ethylene receptors in plants are thought to have been acquired following the endosymbiotic event that led to chloroplasts. In plants, ethylene functions by binding to ethylene receptors to elicit downstream responses. Analysis of cyanobacteria genomes shows that many have proteins with putative ethylene-binding domains with sequences similar to ethylene receptors in plants. Additionally, many cyanobacteria species, including Synechocystis PCC6803, have high-affinity, saturable ethylene binding activity. In this study, we seek to elucidate the biochemical nature of ethylene binding in Synechocystis and determine the physiological role of ethylene in this organism. In Synechocystis, slr1212 encodes a multidomain protein, SynETR1, which contains a predicted ethylene binding domain. We conducted radioligand binding assays on an exogenously expressed, truncated version of SynETR1 that contained only the putative ethylene binding domain. Our results demonstrate that this domain binds ethylene with high affinity comparable to that seen in plants. Previous reports showed that SynETR1 functions as a two-component light receptor mediating the phototactic response of Synechocystis. From this, we hypothesized that ethylene regulates phototaxis through SynETR1. We confirmed this by showing that ethylene enhances phototaxis towards white light, while also

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increasing sensitivity to low levels of light. Disruption of slr1212 leads to a significant increase in positive phototaxis compared to wildtype. Using qRT-PCR, we have quantified the transcript abundance of several genes known to function downstream of SynETR1. Results from these experiments show that SynETR1 is a negative regulator of signaling in directional light. Thus, SynETR1 appears to be an ethylene receptor. Currently, we are exploring the exact physiological output of SynETR1 with preliminary results indicating a possible role in regulation of pili function.

40. Expression and phosphorylation of NMDA receptor subunits in the mammalian suprachiasmatic nucleus during EtOH tolerance and withdrawal Jonathan H. Lindsay1, J. David Glass2, Rebecca A. Prosser1 1 Dept. Biochemistry & Cellular & Molecular Biology, Univ. of Tennessee, Knoxville; 2Dept. Biological Sciences, Kent State Univ.

Alcohol (EtOH) abuse is linked to cancer, mood disorders, and sleep disturbances. The strong link between sleep, circadian rhythms, and alcohol abuse has led us to investigate direct effects of ethanol on the circadian clock located in the suprachiasmatic nucleus (SCN). We have shown that acute ethanol blocks photic phase shifts in vivo and glutamatergic phase shifts in vitro (Prosser et al, 2008). However, neural systems become tolerant to ethanol across acute, rapid and chronic timeframes. Our research has identified acute, rapid, and chronic forms of tolerance in the circadian clock, as well as withdrawal-associated glutamate hypersensitivity (Prosser & Glass 2009; Lindsay et al 2014a,b,c). For rapid and chronic tolerance experiments, adult C57BL/6J mice were given access to 15% ethanol/water solution over varying periods of time depending on the tolerance model. SCN brain slices were prepared and maintained in a perfused brain slice chamber for 24-36 hours. Brain slices were treated at zeitgeber time (ZT) 16 (where ZT 0 = lights-on and ZT 12 = lights off) with glutamate (1mM) + ethanol (20mM) for 10 min or left untreated.

Brain slices were either collected for protein expression assays, or maintained until the following day, when spontaneous neuronal activity was recorded to determine circadian clock phase. Of paramount importance is to determine how the cellular effects underlying the different forms of ethanol tolerance and withdrawal differ. Thus far, we have shown that the NMDA receptor NR2B subunit total expression and NR2B phosphorylation at Tyr 1472 do not change during rapid tolerance to ethanol (Lindsay et al 2014). In contrast, NR2B total expression increases during withdrawal from ethanol, and Tyr 1472 phosphorylation is increased during both chronic tolerance and withdrawal (Lindsay et al 2014c). Here we are extending our investigation to assess the total expression and phosphorylation levels of the NR1 and NR2a NMDA receptor subunits across EtOH tolerance and withdrawal from EtOH. We are also assessing surface expression of NR2B using biotinylation, both across the circadian cycle as well as in response to ethanol consumption. Our baseline experiment suggests that surface expression of NR2B is highest at ZT16 and lowest at ZT23. A better understanding of the dynamics of the NMDA receptors across different forms of EtOH tolerance may lead to novel pharmacological treatment for alcoholism. Support: RO1 AA017898

43. Sphingosine-1-phosphate receptor 1 expression in the suprachiasmatic nucleus Christopher Mendoza, Jonathan Lindsay, Rebecca A. Prosser Department of Biochemistry & Cellular and Molecular Biology Mammalian circadian rhythms are controlled by the molecular clock located in the suprachiasmatic nucleus (SCN).

The SCN circadian clock coordinates daily behavioral and physiological rhythms to synchronize with the external environment. Synchronization primarily involves light signals acting on SCN neurons through release of the neurotransmitter, glutamate, which acts on NMDA receptors. Research in our laboratory is exploring cellular mechanisms that modulate NMDA receptor signaling in the SCN. Previous studies show that activation of sphingosine-1-phosphate receptor 1 (S1PR1) in the hippocampus increases phosphorylation and recruitment of NMDA receptors to the neural membrane; two processes that prime neurons for further excitation. Given what is known about the SCN, we hypothesize that S1PR1 activation alters glutamate phase shifts in the circadian clock. In this study we are investigating whether S1PR1 is expressed in the SCN, and whether it alters NMDA receptor phosphorylation and/or localization. SCN brain slices were prepared from adult male mice and maintained in a Hattonstyle brain slice dish, with Earleâ&#x20AC;&#x2122;s Balanced Salt Solution (pH 7.4, 37°C). Slices were collected at various times for immunoblot assays after different experimental treatments. Results indicate that S1PR1 is expressed in the SCN, possibly at higher levels at night than in the day. Additional experiments are currently investigating whether glutamate stimulation affects S1PR1 expression, and whether activating S1PR1 with the S1P analog FTY720P alters glutamate phase resetting of the SCN circadian clock.

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44. Azospirillum brasilense chemotaxis depends on two signaling pathways regulating distinct motility parameters Tanmoy Mukherjee1, Dhivya Kumar1, Nathan Burriss1, Zhihong Xie1, 2 and Gladys Alexandre1 1 Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA 2 Yantai Institute of Coastal Zone Research, CAS, Yantai, Shandong, P.R. China Bacterial chemotaxis is a key survival strategy in diverse environments. It is also an important behavior that allows soil motile bacteria to colonize plant roots. Most motile bacteria encode two or more chemotaxis (Che) systems in their genome. The function that multiple Che systems play in bacteria has been characterized in very few model organisms. Azospirillum brasilense are motile diazotrophic bacteria that possess four chemotaxis pathways. During chemotaxis, A. brasilense transiently increase swimming speed and suppress changes in the swimming direction, a behavior that allow them to navigate away or toward effectors. One of these chemotaxis operons named Che1 was shown in previous work to regulate transient changes in swimming velocity but which of the other three Che systems regulate the changes in the swimming direction was not known. Using genetic and behavioral assays, we demonstrate that the Che4 chemotaxis system regulates the swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization in A. brasilense. We also show that Che1 and Che4 function together to coordinate changes in the swimming motility pattern and that the effect of Che1 on swimming speed functions to enhance the chemotactic response. In conclusion, our results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis systems to regulate speed and reversal frequency and enhance the chemotactic advantage.

45. Regulation of Pdf transcription in Drosophila melanogaster Sudershana Nair1, Jae Hoon Bahn1 and Jae H. Park1 1 Laboratory of Neurogenetics, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, United States of America Circadian clocks control daily rhythms in physiology and behavior of organisms allowing it to predict environmental changes. Circadian oscillations of various core-clock molecules within pacemaker cells generate and maintain these rhythms. In Drosophila melanogaster, Pigment dispersing factor (PDF) is the major neuropeptide that is produced by central clock cells called latero-ventral neurons (LNvs). PDF synchronizes other clock neurons thereby playing an essential role in the maintenance of circadian rhythms. Core clock transcription factors have been shown to regulate Pdf expression indirectly in the LNvs. However, the underlying molecular mechanism of restricted Pdf expression in the lateral neurons is little understood. Here, using Pdf promoter-bashing experiment, we have identified cis-acting Pdf regulatory element (PRE) that is sufficient for driving Pdf expression in the LNvs. We have further identified a homeobox transcription factor, scarecrow (SCRO) as a PRE binding factor. Transgenic expression of scro in the clock neurons abolished Pdf expression and circadian locomotor activity rhythms. Such repression of Pdf requires DNAbinding homeodomain of SCRO, but not other conserved domains. These results suggest that scro encodes a novel transcriptional regulator repressing Pdf expression in non-PDF expressing clock neurons. SCRO is a member of NK2 homeobox protein family and this is the first report describing its functional significance linked to the biological clock in Drosophila. Our findings can be used to study clock-associated roles for SCRO homolog Nkx2.1 in the vertebrate clock controlling system.

46. Effect of osmotic stress on the folate pathway Deepika Nambiar, Robert Shew, Bryan Schwarz, Michael Duff, Timkhite Kulu-Berhane and Elizabeth Howell Department of Biochemistry Cellular and Molecular Biology, University of Tennessee Knoxville, TN 379976 Folate (Vitamin B9) is involved in one carbon transfer reactions required for the synthesis of important macromolecules such as DNA and amino acids. Since humans cannot synthesize folate, malfunctioning of the enzymes involved in folate metabolism can lead to various diseases like megaloblastic anemia, birth defects, increased risk of cardiovascular disease and cancer. In addition, enzymes in the folate biosynthesis pathway are potential drug targets in bacteria. Our current understanding of the folate pathway is mostly based on in vitro studies, which are very different from the crowded environment that exists in the cell. E. coli is known to produce osmoprotectants, such as glycine betaine, during times of osmotic stress. This leads to perturbation of water activity inside the cell, and an increase in macromolecular crowding. We have shown earlier that, in vitro, osmolytes weaken the binding of dihydrofolate to dihydrofolate reductase of the folate pathway. We hypothesize that an increased osmolyte concentration in the cell will also prevent the functioning of other folate pathway enzymes by interaction of osmolytes with the various folate redox states. Here we studied the effect of osmolytes on the enzymes methylenetetrahydrofolate reductase (metF) and serine hydroxymethyltransferase (glyA) required for methionine and glycine synthesis respectively in E. coli and also dihydropteroate synthase (folP) responsible for biosynthesis of dihydrofolate. Studies were done with knockout and

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rescued strains for the metF, glyA and folP genes. The knockout mutants were restored to prototrophy by addition of folate end products while the rescued strains contain a pKTS plasmid containing the knocked out gene under tetracycline control. The gene is fused with a sequence encoding a SsrA degradation tag to limit the amounts of protein produced in the cell. Osmotic stress studies for metF and glyA indicated that the rescued strain was unable to grow in higher osmolality conditions when compared to knockout strains. We predict this is due to an increase in osmolyte concentration in vivo which leads to interaction of osmolytes with folate intermediates in the pathway. This is turn decreases the efficiency of the folate pathway enzyme

47. A Novel Soluble Peptide with pH-Responsive Membrane Insertion Vanessa P. Nguyen, Daiane S. Alves, Haden L. Scott, Forrest L. Davis, and Francisco N. Barrera Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee - Knoxville, 1414 Cumberland Avenue. Knoxville, TN 37996 Targeted therapies are commonly used for cancer treatments. However, successful targeting of specific molecular markers is limited by tumor heterogeneity. Therefore, it is preferred to target an intrinsic property, such as acidity, instead. The acidic microenvironments in tumors arise from heightened cellular metabolic activity (Warburg effect), which decreases the pH of the tumor environment by increased production of anaerobic metabolites. Acidosis can be a useful biomarker for therapy if methods can be found to target tissue acidity. Here, we show that it is possible to rationally design a soluble peptide (ATRAM) that selectively targets membranes in acidic environments. Fluorescence spectroscopy and circular dichroism were used to study the membrane interaction of the peptide, while confocal microscopy was performed to determine the pH-dependent interactions with in cell lines. The biophysical studies demonstrated that the ATRAM peptide’s membrane interaction is pH-dependent as it binds to the surface of lipid membrane at alkaline pH but inserts into the membrane as an α-helix at acidic pH. The insertion pK of ATRAM into membranes is 6.5, which is similar to the extracellular pH found in solid tumors. Additionally, the peptide targets acidic tumors with high specificity and sensitivity. The pH-responsive property makes the ATRAM peptide an appropriate mean to target tumorous cancer and, potentially, other diseases with altered pH environments.

48. C-di-GMP Mediates Aerotaxis and Receptor Sensitivity Lindsey O’Neal, Min-Hyung Ryu*, Mark Gomelsky*, Gladys Alexandre University of Tennessee, Knoxville; *University of Wyoming Azospirillum brasilense are motile proteobacteria capable of movement in oxygen gradients (aerotaxis). Aerotaxis is the strongest behavioral response in this species which can be observed by the formation of a sharp arerotactic band at some distance from the meniscus when cells are exposed to a spatial gradient of oxygen. Aerotaxis, including aerotactic band formation, is mediated by several chemotaxis receptors including Tlp1. Tlp1 is a prototypical transmembrane chemotaxis receptor which also possesses a c-di-GMP effector domain (PilZ) at the extreme Cterminus. We have previously shown that c-di-GMP binding to the PilZ domain of Tlp1 affected the sensitivity of the receptor and impaired aerotaxis. However, the physiological significance for c-di-GMP regulating a chemotaxis receptor’s sensitivity is not known. Here, we developed an optogenetic tool for the real-time manipulation of intracellular c-di-GMP metabolism in swimming bacteria, and we used it to characterize the role of c-di-GMP signaling during aerotaxis in A. brasilense. First, we showed that this optogenetic system allows manipulation of cdi-GMP metabolism at time scales relevant to chemotaxis since it mediates immediate, yet transient increases or decreases in c-di-GMP content, since the effects dissipate within 10 minutes. Consistent with this observation, transient increases in intracellular c-di-GMP concentrations caused an increase in cell-cell clumping, which is associated with elevated c-di-GMP in A. brasilense. No increase in clumping was observed in a mutant lacking tlp1 under similar conditions, providing further evidence for a link to chemotaxis signaling. Second, we demonstrated that c-di-GMP itself is not a cue sensed by aerotaxis receptors since increasing c-di-GMP or decreasing c-di-GMP content of free-swimming cells did not affect their motility patterns. Using a mutant altered in c-di-GMP metabolism and additional aerotaxis assays, we also provide evidence that c-di-GMP likely regulates the sensitivity of most, if not all, receptors that contribute to the formation of a sharp aerotactic band in A. brasilense, suggesting a function for three other PilZ-containing receptors. Last we provide evidence that c-di-GMP binding to receptors has a direct role in allowing cells to form a sharp aerotactic band and we further suggest that c-di-GMP binding to receptors fine-tunes their sensitivity to a narrow range of oxygen concentration to navigate cells toward a precise low oxygen concentration in a gradient.

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51. DOMINANCE OF OLD END GROWTH IS INHERITED IN FISSION YEAST Julie Rich, Bin Wei, and Maitreyi Das Department of Biochemistry & Cellular & Molecular Biology Cells in all organisms must attain an appropriate shape in order to function properly. Significant research has been done to describe polarized cell growth, but the mechanism by which a cell breaks symmetry is unknown. Previous studies in cylindrical fission yeast cells show that the old end and the new end compete with each other for active Cdc42, which is the primary regulator of eukaryotic polarized cell growth; the old end, which is the dominant end, wins initially. Gef1 is an activator of Cdc42, and gef1Δ cells display unique growth patterns comprised of cells that grow in either a bipolar or monopolar manner. We aimed to discover what imparts old end dominance by characterizing cell growth patterns and Cdc42 dynamics in wild-type and gef1Δ mutants via cell image analysis. Our data suggests that the old end is dominant only if it grew in the previous generation, indicating the existence of a physical memory of growth that determines dominance and is inherited through generations. Further studies will strive to identify the factor that imparts this memory of growth and to explain the mechanism by which it imparts dominance, thus getting us closer to understanding how a cell breaks symmetry.

52. Nitrergic signaling in the inferior colliculus Andrew Stafford and Dr. Jim Hall Dept. Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, 37996 astaffo5@utk.edu, jhall1@utk.edu Nitric Oxide (NO) is a gaseous molecule that functions as a retrograde messenger. Activation of glutamate N-methylD-aspartate (NMDA) receptors stimulates NO production via the activity of nitric oxide synthase (NOS). NO is released and subsequently enhances the presynaptic release of glutamate. Staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) have revealed the presence of NOS-labeled neurons in a number of vertebrate brain structures including the inferior colliculus (IC), an important auditory processing center. However, the function of NO in auditory processing at the level of the IC is not known. Here we address this issue using NADPH-d histochemistry, NO microsensors, and single-unit recording combined with microiontophoresis to investigate the role of NO in the analysis of acoustic signals by neurons in the IC of the northern leopard frog, Rana pipiens. Utilizing a slice preparation of the frog’s IC (n=4), bath application of glutamate (10-100 mM) elicited a concentration-dependent release of NO (69.4–273.1 nM). The increase in NO generation induced by glutamate was blocked by the glutamate receptor antagonist, AP5 (200 uM). In vivo iontophoretic application of L-NAME (a NOS inhibitor) was used to evaluate the effect of NO on the sound-evoked responses of neurons (n=4) in the IC. No change in discharge pattern, best frequency, threshold, or tuning curve of either phasic or tonic units was seen during LNAME application. However, the neurons display a greater response specificity for bullfrog calls compared to other frog species’ calls in the presence of L-NAME. Our data suggest a role for NO in gain control in the IC that may influence the output of neural circuits engaged in the analysis of behaviorally relevant acoustic signals.

53. Defining the role of insulator proteins in DNA replication and cell division Emily Stow, Ran An, Mariano Labrador Department of Biochemistry & Cellular & Molecular Biology Nuclear organization is essential for the regulation of gene expression and replication of the genome. This organization is partly mediated by the binding of insulator proteins to insulator sites throughout the genome. Chromatin insulators contribute to the three-dimensional organization of the genome by stabilizing long-range interactions between distant genomic sites along the chromatin fiber. Classic properties of chromatin insulators include their ability to define chromatin boundaries by preventing the spread of heterochromatin and by blocking enhancer-promoter interactions when located between enhancers and promoters. Insulators contribute to transcriptional regulation by promoting contacts between enhancers and regulatory regions of genes. Recent studies show insulator proteins and DNA replication-related factors bind similar genomic regions and that Drosophila insulator, Supressor of Hairy Wing [Su(Hw)], co-immunoprecipitates with proteins involved in establishing origins of replication. These studies link insulators with DNA replication, but provide no details about the precise mechanism of insulator activity during replication. In this study, we analyze the relationship between Su(Hw) and a newly identified insulator partner protein, Heterochromatin and Insulator Partner Protein 1 (HIPP1), in order to strengthen the connection between insulator proteins and DNA replication and to propose a mechanism by which Su(Hw) and HIPP1 mediate the release of Su(Hw) contacts during replication. We use transgenic flies and immunostaining of polytene chromosomes to better understand the relationship between Su(Hw) and HIPP1 and their activity during DNA replication. Our results show that HIPP1 has the ability to release Su(Hw) insulator activity and that these proteins play active roles in regulating the cell cycle.

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54. Roxyâ&#x20AC;?7: a novel peptide that activates the EphA2 receptor.

Justin Westerfield*1, Daiane S. Alves*1, Jennifer L. Morrell-Falvey2, and Francisco N. Barrera1 1 Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 1414 Cumberland Avenue. Knoxville, TN 37996.

2 Biological and Nanoscale Systems BioSciences Division ORNL, Oak Ridge, TN 37831-6445 *These authors contributed equally to this work.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptor is involved in several key processes such as cell proliferation, migration, differentiation and apoptosis, which help to maintain cell cycle homeostasis.

Dysregulation of these cellular processes perturbs the tight physiological control of healthy cells and can give rise to the pathological states implicated in the development of cancer. A key receptor for these processes in humans, EphA2, is overexpressed in several cancers with poor outcomes. The differential expression of EphA2 in some cancer types compared to normal cells highlights its importance as a therapeutic target. Despite its overexpression, multiple studies have documented low levels of EphA2 phosphorylation in malignant cells. In the present work, we designed a peptide (Roxy-7) that targets the EphA2 receptor and significantly increases EphA2 phosphorylation (p-Tyr) in non-small cell lung cancer (NSCLC) in vitro. Roxy-7 is soluble until acidity triggers its insertion into lipid bilayers and adoption of Îą-helical secondary structure, highlighting a potential targeting mechanism. Structured Illumination Microscopy (SIM) experiments indicate that Roxy-7 enhances endogenous EphA2 receptor clustering at the plasma membrane.

We also tested the toxicity of the peptide in cancer cells by MTS assay and the results show that Roxy-7 is not toxic.

Finally and most importantly, Roxy-7 inhibits cell migration in two different cancer cell line models suggesting that Roxy-7 has potential for metastasis treatment. In summary, here we investigate a new potential therapeutic molecule that enhances phosphorylation of EphA2 receptor and inhibits cell migration.

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Chemical and Biomolecular Engineering Student Abstracts 55. Distinct locations of lipid droplet biogenesis on the endoplasmic reticulum are driven by neutral lipid content Zuania Pacheco del RĂ­o1, Alex Meyers1, Paul Dalhaimer123 1 Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN 37996-2200, USA 2 Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA 3 Institute of Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, USA Obesity is one of the most widespread diseases in the United States and the world, and it has been related to several health complications; increase on blood pressure, cholesterol levels, heart coronary disease, strokes, type 2 diabetes, and a higher probability of certain types of cancer. Obesity is a consequence of an increased amount of fat in the body, an excess on the quantity of neutral lipids stored in organelles called lipid droplets. Our goal is to determine how lipid droplets form from the endoplasmic reticulum (ER) in order to improve the understanding of the cellular origins of obesity.

We used the yeast Schizosaccharomyces pombe (S. pombe), where the nuclear and cortical/peripheral ER domains are easily distinguishable by light microscopy, and a S. pombe mutant CDC25, which can be arrested at mitotic stage g2, where the majority of lipid droplets are form. An unusual lipid droplet formation behavior was observed when S. pombe cells are arrested in mitotic stage g2, lipids droplets were forming close to the peripheral ER. We hypothesized that this droplets had a high content of sterol esters (SE) since localized droplet formation can be explained by the presence of sterols in neighboring, growing regions of the cell. Chromatography analysis confirmed an increase in the amount of SEs in CDC25 cells arrested at mitotic stage g2, while an increase in the amount of triacylglycerol (TAG) in wild type S. pombe when lipid droplets where forming at the nuclear ER was observed. We notice that TAGs droplet formation around the nuclear ER appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which is localized exclusively to the nuclear ER, and Dgk1p, which is localized strongly to the nuclear ER over the cortical/peripheral ER. So far we had found sufficient data to believe that the lipids droplets that are being formed in the peripheral ER are mostly composed of SEs, while the lipid droplets formed at the nuclear ER are mostly composed of TAGs. These results profoundly modify the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis site in the ER.

Chemistry Student Abstracts 56. Enhancing the Cyanoacrylate Fuming Method of Latent Prints by Optimizing Temperature and Humidity 1 Leondra S. Lawson, 1Christina Jackson, 1Mark Dadmun 1 Department of Chemistry, University of Tennessee, Knoxville, TN The cyanoacrylate (commonly known as superglue) fuming method (CFM) is a widespread chemical process used in forensics to reveal latent prints on surfaces via the anionic polymerization of ethyl cyanoacrylate (ECA) with biological components, such as the amino acids found in sweat, which serve as initiators.. Although this method is frequently used, studies to-date have failed to identify the chemical processes that occur at a molecular level. Empirical studies have shown that the polymerization of ECA at room temperature produces the most polymer when the relative humidity is around 80 percent. Recent studies in the Dadmun group have also shown that the anionic polymerization of ECA at low temperatures produces a larger quantity of high molecular weight polymer. Although the exclusive effects of relative humidity and temperature on the growth of poly(ethyl cyanoacrylate) (PECA) from deposited prints during fuming have been thoroughly investigated, our research exceeds the scope of current knowledge by evaluating the collaborative influence these parameters have on the method. To test the combined effects of humidity and temperature on the efficiency of the CFM, latent fingerprints on glass slides were placed in a fuming tank and fumed at different humidity levels and temperature settings. The amount of polymer accumulation and quality of the prints were compared. Our data supports previous findings in regards to lower temperatures and higher relative humidity producing higher accumulation of PECA. Further studies are required to determine if higher mass of PECA at lower temperatures is the result of condensation on the slides due to the excessive moisture present in the system. This holistic study provides forensic scientists with more optimum procedures to obtain fingerprints from surfaces without deteriorating the evidence in the process.

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Comparative and Experimental Medicine Student Abstracts 57. Antimicrobial Resistant Staphylococcal Infections in Horses in Kentucky Ronita Adams, Department of Comparative & Experimental Medicine, University of Tennessee Co-authors: Dr. Agricola Odoi University of Tennessee Department of Biomedical and Diagnostic Sciences (Advisor), Dr. Jackie Smith University of Kentucky Veterinary Diagnostic Laboratory, and Dr. Craig Carter University of Kentucky Veterinary Diagnostic Laboratory Antimicrobial resistant staphylococcal infections are becoming a growing concern in the field of equine medicine.

Moreover, these infections are of public health concern because of evidence of cross transmission between humans and animals. Understanding the geographical distribution of these infections is important in identifying communities at higher risk of the infections and hence in identifying factors responsible. However, little is known about the geographic pattern of equine antimicrobial resistant staphylococcal infections. Past studies fail to address the proportion and pattern of antimicrobial resistant staphylococcal infections in the general equine population in the United States overall and Kentucky, in particular. In order to address this issue, an investigation of the geographic patterns of equine antimicrobial-resistant staphylococcal infections in Kentucky needs to occur. Therefore, the objective of this study is to use diagnostic laboratory data, specifically on antimicrobial resistance from horses in Kentucky to investigate spatial patterns of equine antimicrobial drug resistant staphylococcus infections in Kentucky.

Data preparation/cleaning and statistical analysis will be done using SAS. ArcGISwill be used to generate choropleth maps as a means of visualizing the patterns of disease. The results of the visualization are expected to allow identification of distributions or clustering of antimicrobial resistant staphylococcal infections in the population that are more likely in specific locations of the study area.

Ecology & Evolutionary Biology Student Abstracts 58. Revisiting the ecomorph classification system of anole lizards using three dimensional territory estimates Jordan Bush, Daniel Simberloff Department of Ecology & Evolutionary Biology Anole lizards (genus: Anolis) escape competition by specializing in different microhabitats along tree trunks, an adaptive radiation that has resulted in over 400 speciation events. Because of this phenomenon, anoles are generally classified into “ecomorph” categories based on their morphology, behavior, and preferred microhabitat. Yet it is common to see anoles outside of their expected perch heights, and little is known about how these species behave or communicate in three dimensions. In this study, we created a method of estimating territory size and shape in three dimensions to test how well Cuban brown anoles (Anolsi sagrei) behaviorally conform to their ecomorph classification. We predicted that a high incidence of brown anoles would have territories outside of their expected “trunk-ground” range, and that territory volumes will be better predictors of display behaviors than other territory metrics. To test this, we estimated the area and volume of male territories in a natural population of brown anoles and compared these metrics to individuals’ display behaviors and social network. We found that display rates were not related to any estimate of territory size, and that the majority of brown anoles had territory estimates within their expected height range. This indicates that the traditional ecomorph system is representative of the habitat use of this species. The results of this study and the techniques it introduces pave the way towards more fully understanding anole social behavior in the context of ecomorph categories, and ensures that these model reptile species continue to be represented accurately by their classification systems.

59. Eutrophication in Marine Environments: How the Addition of Excess Nutrients Contributes to Shifts Toward Algae Dominance Domonique Hatton1 Camille Gaynus2 Christian Henry3 Keeli Howard3 Melissa Kemp4 Peggy Fong2 Gladys Alexandre1 1 University of Tennessee 2 \ University of California Los-Angeles 3Hampton University College 5Standford University Humans have relied on oceans and other large bodies of waters for a myriad of reasons to include using them a source of food, for waste dump, and for recreational purposes. Not until recently have people come understand the ways that humans impact oceans negatively. The health of coral reefs and balance of systems along marine shelfs are a major concern. As coral reefs are destroyed faster than they can repair themselves the occurrence of large algae blooms increases. Research conducted in marine systems has brought awareness to anthropogenic activities to include; over

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fishing, eutrophication, and pollution. Pollution in water systems and over fishing have been studied and mechanisms to explain the effects they have been developed while the process of eutrophication and all the possible effects it could have on marine environments have not been investigated to the same extent. Eutrophication, a unique form of pollution, occurs when excess nutrients are released into water systems. We aim to understand how this disturbance in marine environments, that are normally nutrient poor and have tight nutrient cycles, can lead to shifts in marine environments towards algal dominance. In a series of cafeteria style choice experiments we use herbivorous fish of the shore lines of beaches in Moore, French Polynesia to find which species of macroalgae (Galaxaura, Sargassum, and Padina) are more palatable. The next choice experiments tested two treatments of the algae nutrient- enriched and non-enriched algae. We find evidence that that excess nutrients can change the palatability of algae. The Padina which was consumed the most in the first tests was eaten significantly less when treated with nutrients. We believe that with access to more nutrients macroalgae can allocate more energy for defenses to make them less palatable to herbivores. Understanding how different species of macro algae allocate resources for chemical and structural defenses may be crucial for treating and restoring coral reefs around the world.

60. Threeâ&#x20AC;&#x2122;s company: Microbial interactions and chemical defenses within myrmecochory Chloe Lash and Charles Kwit Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996 Animal-mediated seed dispersal, a plant-animal mutualism, is prominent in many natural systems and plays important roles in plant community structure, population dynamics, evolution, and animal diet. Studies of mutualistic seed dispersal generally focus on proposed beneficial outcomes to the two main parties, such as distance or microsites away from a parent plant for seeds or a nutritious food source for animals. Little attention, however, has been given to chemical components of these mutualisms and how they may interact with third parties, particularly microbes, present in the environment. To study the chemical components of animal mediated seed dispersal, myrmecochory (ant seed dispersal) offers an ideal system. Ant metapleural gland secretions, which have antimicrobial properties, will be tested for their inhibitory effects on seed coat-associated microbial growth. Additionally, plant antimicrobials, such as sanguinarine in Sanguinaria canadensis, will be tested for effects on ant nest-associated microbes and ant colony survival. We expect these chemical components to inhibit some microbial growth, which would indicate that a microbial third party may be complicating what is normally assumed to be a simple pairwise mutualism. The results of this project could provide insight into the role of chemicals in other animal-mediated seed dispersal mutualisms, including those involving chemical seed treatment in frugivore guts.

61. An Agent-Based Model of Golden Eagle Predation on the Santa Cruz Island Fox Shelby M. Scott1 and Erin N. Bodine2 1. Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 2. Department of Mathematics and Computer Science, Rhodes College, Memphis, TN During the 1990s, the endemic Santa Cruz island fox (Uroycyon littoralis santacruzae) population experienced heavy predation from invasive golden eagles (Aquila chrysaetos), which had recently migrated to the island. Predation from the golden eagles, in combination with other factors, nearly caused the Santa Cruz island fox population to become extinct. Previous mathematical models of this predator-prey system have made limiting assumptions of this system. We present an agent-based model (ABM), which simulates the population dynamics of the Santa Cruz island fox population as well as the predation by golden eagles. Geographic information system (GIS) data are integrated into the model to represent the distribution of various types of vegetation on the island. The data also give the location of golden eagles nests and hunting territories. Using the model, we determine how both island vegetation and predation by golden eagles impact the spatial density and distribution of the foxes across Santa Cruz island. We also observe that as few as ten golden eagles could have caused this rapid population decline. This research gives insight to the antagonistic relationship between golden eagles and foxes and can be used to rescue other fragile island communities.

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Microbiology Student Abstracts 62. Host range study of a lysogenic phage in a roseophage-roseobacter system and characterization of lysogens in affecting marine biogeochemistry J. BASSO, M. TUTTLE, A. GROSSMAN, N. ANKRAH, A. BUCHAN. Department of Microbiology, University of Tennessee-Knoxville, Knoxville, TN Viruses are abundant in the marine environment, where they actively infect marine bacteria and contribute to biogeochemical cycles. Bacterial viruses (phage) display two distinct lifestyles: host lysis or integration in their host’s genome (where they are termed prophage). Prophages lie dormant until environmental conditions trigger their induction to a lytic cycle. In a limited number of well-studied phage-host systems, prophages have been able to provide their hosts immunity against infection by other phage. The extent to which this immunity is prevalent in marine bacteria and influences the occurrence of lytic events has not been well studied. We have isolated a marine bacterium, Sulfitobacter sp. CB2047, and its infecting phage (ϕCB2047-A). The bacterium contains a prophage with high sequence similarity (85.65% identity) to ϕCB2047-A. We have also isolated four additional Sulfitobacter strains, isolated from distinct environments and none of which contain a prophage. Integration experiments of Sulfitobacter CB2047 with ϕCB2047-A results in a substitution of the prophage, yield the strain YM3A. We hypothesize that phage integration is possible in the additional Sulfitobacter strains and have data to support this for Sulfitobacter sp. EE-36.

These data increase our understanding of the biotic factors that stimulate prophage integration. We also hypothesize that prophage in their lysogenic state will affect host physiology. Indeed CB2047 and YM3A have distinctly different phenotypes with respect to growth rates, cell size, and biofilm formation, suggesting that roseophage have the ability to dramatically alter bacterial physiology and marine biogeochemistry.

Public Health Student Abstracts 63. Social Aspects of Adolescent Non-Medical Use of Prescription Drugs Michelle J. Smith1, Laurie L. Meschke1, Laura Miller2, Jonathan Pettigrew2, Clea McNeely1 1Department of Public Health, University of Tennessee, Knoxville 2 School of Communication, University of Tennessee, Knoxville Non-medical use of prescription drugs (NMUPD) is an person’s use of a medication that was prescribed to someone else or the use of a personal prescription in a way or dosage outside of the original purpose (National Institute for Drug Abuse (NIDA), 2012). In 2005 2.1 million teens abused prescription drugs, according to the Office of National Drug Control Policy (ONDCP, 2007), with an estimated 850,000 12-17 year old adolescents initiating NMUPD. As a result, prescription drugs are now the second most abused illegal drug among adolescents (ONDCP, 2007), exceeded only by marijuana. A comprehensive understanding of current research associated with adolescent NMUPD is critical in developing research-based interventions. We address this need within the context of the ecological model (Bronfenbrenner, 1988, 1989), examining factors related to NMPD at the individual, family, peer, and community levels.

We searched numerous social science and medical databases (e.g., Academic Search Premier, CINAHL, MEDLINE, Psychology and Behavioral Sciences Collection, PsycINFO, and Sociological Collection) using consistent keyword searches (e.g., "prescription drug abuse"). This systematic review identified 35 peer-reviewed published manuscripts that used quantitative and/or qualitative methods. The variables or phenomenon included in the studies were then analyzed in relation to the ecological model (Bronfenbrenner, 1988, 1989). Specifically we identified risk and protective factors (n=97) related to adolescent NMUPD and categorized these into individual (n=61), family (n=13), peer (n=10), or community (n=13) levels.

Preliminary findings reveal an emphasis on the individual level factors in the current research —59 of the 95 measures (62%) reflected individual factors. The remaining factors were evenly distributed across family (12 measures), peer (10 measures), and community (14 measures) levels. The vast majority of the measures (n = 61; 64%) were included in only one of the 35 articles reviewed so replication of the findings is quite low. Nevertheless, the proportion of significant factors related to NMUPD was high: 79% at the individual level; 58% at the family level; 90% at the peer level; and 57% at the community level. Significant risk factors at the various levels included: other substance use, sexual assault, peer delinquency, foster care, and rurality.

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2016 CBP Poster Competition Judges We would like to thank our 2016 poster judges for your support.

Paul Abraham OgĂźn Adebali Vasilios Alexiades Daiane Alves Josh Bembenek Jeremy Chandler Jiangang Chen Xiaolin Cheng Sung ki Cho Dan Close Judy Day Michael Duff Shigetoshi Eda Vitaly Ganusov Terry Hazen Liz Howell Dan Jacobson

Nels Johnson Juan Luis Jurat-Fuentes Zhou Li Yutao Liu Ansul Lokdarshi Stephanie Madison Barmak Mostofian Estefania Paredes Jerry Parks Dale Pelletier Lenora Pluchino Sangeetha Rajagopalan Pratyush Routray Ioannis Sgouralis Marcy Souza Bin Wei Jason Whitham

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