HHMI-Sponsored UD Undergraduates at the Experimental Biology Meetings, New Orleans 2009

Anaheim Convention Center

EB2010
EXPERIMENTAL BIOLOGY MEETINGS
Anaheim, CA APRIL 24-28, 2010

For the past 10 years, the University of Delaware Howard Hughes Medical Institute’s (HHMI) Undergraduate Science Education program has sent undergraduate students to the Experimental Biology Meetings to present their research. As part of this conference, the American Society for Biochemistry and Molecular Biology sponsored its 14th Undergraduate Poster Competition in which 13 UD students participated. Since 2001, students from the University have received more awards in this competition than students from any other college or university. This year two students, Amy Styer and Laura Sloofman received Honorable Mentions in this competition. See article in November 2008 ASBMB Today and website for last year's meeting in New Orleans.

The University of Delaware group included four faculty and 13 undergraduates.

From left to right: Michael Napolitano, Jamie Stull, Amy Styer, Jean Huynh, Rebecca Brown, Megan Kissig, Laura Sloofman, Katharine Shelly, Tyler Larsen, Tejal Naik, Rachel Randell, Robert Sheehan, and Aleksey Dvorzhinskiy.
Prof. Hal White, Chem & Biochem Prof. Dave Usher, Biol. Sci. Prof. Seung Hong, Biol Sci Prof. Gary Laverty, Biol Sci	Rebecca Brown Aleksey Dvorzhinskiy Jean Huynh Megan Kissig	Tyler Larsen Tejal Naik Michael Napolitano Wachen Peters	R achel Randell Robert Sheehan Katharine Shelly	Laura Sloofman Jamie Stull Amy Styer

University of Delaware students and their abstracts.

Rebecca Brown

MicroRNAs Associated with Environmental Stress inArabidopsis

Rebecca Sul Hee Brown¹, Dong-Hoon Jeong^2,3, Blake C Meyers^2,3, Pamela J Green^2,3
¹Biological Sciences, ²Plant and Soil Sciences, University of Delaware, Newark, DE, ³Delaware Biotechnology Institute, Newark, DE

One way plants respond to environmental stress is by modifying their gene expression through the use of microRNAs (miRNAs). miRNAs are noncoding small RNAs that regulate gene expression at the posttranscriptional level by base pairing with complementary messenger RNA (mRNA) molecules, causing either mRNA cleavage or translational inhibition. To elucidate the roles of miRNAs in environmental stress responses, wild type plants and miRNA enriched mutant plants were subjected to various environmental stresses. Small RNA libraries from stress-treated seedlings and flowers were constructed and sequenced using deep sequencing technologies. Computational analysis revealed differential expression of miRNAs between stress-treated plants and control plants with some miRNAs upregulated and some downreguled by environmental stress. Additional analysis revealed potentially novel miRNAs in Arabidopsis as well. These results suggest that miRNAs play important roles in stress responses, and that miRNA identification in Arabidopsis has not been saturated. Future work will involve validating highly stress-regulated miRNAs and prospective new miRNAs, and examining their potential target genes for regulation. Stress-regulated miRNAs will be incorporated into multi-network models and their biological roles will be hypothesized and tested through functional studies. R.S.H.B. was supported by the Howard Hughes Medical Institute, and NSF grant MCB#0548569 to P.J.G. provided research support.

Abstract Number: 4114

Aleksey Dvorzhinskiy

Recipient of an ASBMB Undergraduate Travel Award.

Efflux pump upregulation in DTAC-
(Dodecyltrimethylammonium Chloride) Resistant Salmonella

Aleksey Dvorzhinskiy, Megan Kautz, and Diane Herson
Department of Biological Sciences

Salmonella spp. are gram-negative pathogens estimated to cause 1.4 million cases of food poisoning annually in the United States and are increasingly becoming resistant to disinfection in a variety of environments. In this study, Salmonella enterica 4931 strains were developed which were able to grow in the presence of 500600 ppm Dodecyltrimethylammonium chloride (DTAC), a quaternary ammonium compound (QAC) and also displayed a cross resistance to penicillin. Later, these strains with reduced susceptibility (SRS) were passaged in the absence of DTAC to mimic environmental conditions after exposure an antimicrobial. The minimum inhibitory concentration (MIC) of DTAC for these strains (DSRS) did not vary appreciably from the SRS level for forty passages. The results from our microarray studies support the findings that reduced susceptibility to QACs is linked to an upregulation in the transcription of acrB, an efflux pump. This study also showed that SRS and DSRS strains in the presence of the efflux pump inhibitor showed an even greater sensitivity to DTAC than the parental strain. The discovery of similar efflux pump inhibitors could lead to advances in combating SRS strains in clinical and natural environments. Supported in part by an HHMI undergraduate research award.

Abstract number: 2838


Jean Huynh

Mammalian sperm are dependent on JAM-A for normal motility: 
progressing from mice to humans

Jean Huynh, Rolands Aravindan, and Patricia A. Martin-DeLeon
Department of Biological Sciences, University of Delaware Newark, DE 19716

Defective sperm motility (asthenozoospermia) is a primary cause of male infertility. Junctional Adhesion Molecule-A (JAM-A) has been recently shown to be essential for mouse sperm motility. However, to date it has not been studied in human sperm. The objective of this study is to determine if JAM-A is present in human sperm and to characterize its expression. In silico analysis reveals 83% similarity in the amino acid sequence of JAM-A in mice and humans, indicating that the proteins are conserved. This suggests that JAM-A may also play a role in human sperm motility. Western analysis of human sperm proteins revealed the presence of JAM-A with a MW of ~32kDa, identical to that of the mouse. Immunocytochemistry was used to localize the expression of JAM-A in human sperm. Flow cytometry confirmed the Western blot data and revealed the presence of the protein on the sperm membrane in one of two individuals attending an infertility clinic. Once JAM-A has been fully characterized in human sperm, the next step will be to determine its interacting protein partners by attempting to co-immunoprecipitate it with candidate proteins. The localization and characterization of JAM-A in human sperm is expected to increase our understanding of genetic factors leading to human male infertility and subfertility, laying the groundwork for diagnosis of a subset of couples with fertility issues. Funded by NIH-COBRE grant #5P20RR015588-07.

Abstract Number: 6236

Megan Kissig

JAM-A regulates the progression 
of Non-alcoholic Fatty Liver Disease (NAFLD)

Megan E. Kissig and Ulhas P. Naik
Department of Biological Sciences

Non-alcoholic fatty liver disease (NAFLD) is characterized by an abnormal amount of fat accumulation in the liver, specifically more than 5% fat by weight. Little is known about how the fat accumulates in the liver, but it has been found that intestinal permeability due to leaky tight junctions may be a contributing factor. Our lab studies junctional adhesion molecule-A (JAM-A), a protein located at the tight junctions of epithelial and endothelial cells. Through its ability to homodimerize at the apical part of the lateral membrane, JAM-A helps regulate the permeability and stability of the junction. This study’s aim was to find what effect JAM-A has on the development of NAFLD. To analyze this relationship, groups of Jam-A (+/+) and Jam-A (-/-) mice were put on either a high-fat or low-fat diet for 20 weeks. During this time the mice were weighed every two weeks and blood samples were taken every four weeks. At the end of the 20 weeks, the mice were sacrificed and the livers and fat pads were removed. We found that the variation in weight between the Jam-A (-/-) groups was greater than that between the Jam-A (+/+) groups. Also, the average area of the adipocytes in the high-fat Jam-A (-/-) group was greater than the average area in the high-fat Jam-A (+/+) group. Since the liver sends fat to tissues in the form of cholesterol, we compared the levels in the plasma. The high-fat Jam-A (-/-) mice had significantly higher LDL-cholesterol and total cholesterol levels in the plasma than the other groups as well. After examining the stained sections of the livers, it was found that the livers of the high-fat Jam-A (-/-) mice showed more fat droplet accumulation than the high-fat Jam-A (+/+) mice. This suggests that ultimately the absence of JAM-A from the tight junctions promotes the development of NAFLD. This project was funded by the Arnold and Mabel Beckman Foundation.

Abstract Number: 1046

Tyler Larsen

Inherantly Antibacterial Hydrogels –
Altering ACctivity Via Tryptophan/Arginine Iinteractions

Tyler Larsen, Daphne A. Salick, Radhika Nagarkar, Joel P. Schneider
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716

Hydrogels are heavily hydrated materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. The development of antibacterial hydrogels has been of great interest to the hydrogel research community as a means to combat the threat of infection during material implantation. We have developed MAX1, a self-assembling b-hairpin peptide hydrogel whose surface exhibits inherent antibacterial activity against several pathogens prevalent in hospital settings. Under physiological conditions, MAX1 self-assembles into a crosslinked, mechanically rigid hydrogel, but the process is too slow for use as an injectable implant. This study aims to design a hydrogel with rapid self-assembly and high rigidity under physiological conditions while maintaining potent inherent antibacterial activity through incorporation of cation-p interactions, which are common in antibacterial peptides found in nature. A new b-hairpin peptide (RWMAX1) was designed, incorporating a cross-strand R/W pair into the MAX1 sequence. The folding and self-assembly properties were assessed using circular dichroism and rheology and the antibacterial activity was investigated against gram positive S. aureus and gram negative E. coli. RWMAX1 gels were found to possess both favorable physical and antimicrobial properties. Supported by an Undergraduate Beckman Award.

Abstract Number 2386

Tejal Naik

ASBMB 2010 Thematic Best Poster selected by the theme organizers for its outstanding research in Chemical Biology and Drug Discovery.

Development of a Peptide Nucleic Acid Based siRNA Delivery System

Tejal U. Naik and Millicent O. Sullivan

Small interfering RNA (siRNA) plays a major role in gene silencing. The ability to harness siRNA for therapeutic benefit can have a widespread impact on a variety of diseases. However, the efficacy of such treatments is limited due to the many barriers associated with siRNA delivery. In this work, we lay the groundwork for the development of a peptide nucleic acid (PNA)-based surface-mediated siRNA delivery system. PNAs are nucleic acid analogs that hybridize with complementary DNA or RNA sequences, enabling the direct attachment of various macromolecules such as peptides. Conjugation of targeting and protective moieties can potentially enhance the delivery of siRNA. We first established a cell transfection model utilizing stably transfected B16FO mouse melanoma cells producing GFP. Anti-GFP siRNA was designed and its efficacy was evaluated via flow cytometry and fluorescence microscopy. Optimization of cell seeding density, siRNA concentration, use of antibiotics, and time of transfections was accomplished. Our second task was to prepare molecular conjugates for PNA-based siRNA modifications. PNA-peptide conjugates were assembled and purified through Reverse Phase HPLC. Future development of this delivery system will include linkage of anti-GFP siRNA to surfaces via the PNA-peptide tethers, and exploration of the delivery system in B16FO cells.

Abstract Number: 6946

Michael Napolitano

An Evolutionary and Functional Analysis

of Vibrio Pathogenicity Island 2

Michael G. Napolitano, Moreno, S. A., Duncan, M., and Boyd, E. F.

Department of Biological Sciences

The evolution and emergence of pathogenic bacteria is tightly linked to the horizontal transfer of pathogenicity islands (PAIs) and bacteriophages encoding a range of virulence factors. We studied the evolutionary relationships between integrases and excisionases from a group of islands related to Vibrio Pathogenicity Island-2 (VPI-2) found in pathogenic Vibrio cholerae isolates and used VPI-2 as a model for pathogenicity island excision and integration. We determined the functional role of the VPI-2-encoded integrase (intVPI-2) and two putative excisionases (vefA and vefB) in excision. We found negligible levels of excision as measured by attB levels in the ΔintVPI-2 strain whereas overexpression of intVPI-2 showed an increase in excision. In the ΔvefA excision is severely decreased. Overexpression of vefA (VC1785) in the wildtype causes complete excision of the island, while complimenting vefA in either of the deleted strains ΔvefA and ΔvefB leads to only a minor increase in excision. Overexpression of vefA also causes excision of a second island, VPI, critical for pathogenicity. For the first time, we have demonstrated that a factor encoded on one island, vefA, causes the excision of a second. Supported by an Undergraduate Beckman Award.

Abstract Number: 1976

Wachen Peters

P2 Receptors and ATP Enhance the Migration of Prostate Cancer Cells

Wachen Peters, Christine Maguire², Randall L. Duncan², and Robert A. Sikes²

Lincoln University¹ and University of Delaware²

Purinergic signaling stimulates many biological processes. Two classes of purinergic receptors, GPCR (P2Y) and gated channels (P2X), have been identified that bind ATP as a ligand, which can promote cell migration, a critical component of metastasis. ATP also has been shown to have an anticancer effect in vivo. The purpose of this study was to ascertain the effect of ATP on the migration of an isogenic progression series of prostate cancer (PCa) cell lines on three different extracellular matrices. We hypothesize that ATP treatment activates purinergic receptors that increases cell migration on all three extracellular matrices (ECM). RT-PCR was used to determine the mRNA expression profile of P2 receptors and ecto-nucleotidases. Migration was examined using wound healing assays as followed by photo microscopy for four days, as well as RT-PCR to determine effect of interaction with each ECM on the mRNA profile. ATP increased the migration of LNCaP cells on Collagen I to a higher degree than on both Schwann and matrigel over vehicle alone. Peak wound closure occurred after 48 hours. Differential expression of P2 receptors and ectonucleotidases was seen when cells were grown on different matrices. ATP is necessary for PCa migration on collagen I, matrigel and Schwann cell matrix. The differential expression of P2 receptors is speculated to be responsible for variable wound healing on extracellular matrices.

Funded by: Department of Defense HBCU/MI Undergraduate Research Training Grant, PC080950; NIH INBRE P20RR016472, NIH COBRE P20 RR015588

Abstract Number: 1053

Rachel Randell

Heparan and chondroitin sulfate modifications in signaling

pathways regulating articular cartilage homeostasis

Rachel Leigh Randell, Richard Wittmeyer, and Erica M. Selva
Department of Biological Sciences, University of Delaware, Newark, DE 19716

Degradation of articular cartilage leads to osteoarthritis (OA) in humans. Articular cartilage homeostasis depends on signaling events modulated by extracellular matrix glycoproteins. We tested the hypothesis that disrupting heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans would alter signaling pathways relevant to cartilage homeostasis. Mosaic mutant and wild-type Drosophila wing discs were used to visualize the effects of mutations in two genes: O-xylosyltransferase (oxt), required for linking HS and CS to core proteins, and sulfateless (sfl), essential for decorating HS with negatively charged sulfate groups. We performed immunostaining of signaling ligands and pathway activation targets, including: Decapentaplegic, the homolog of human Bone Morphogenic Protein involved in anabolic cartilage processes; Wingless, the homolog of human Wnts implicated in cartilage repair; and Hedgehog and its receptor Patched. We demonstrate the importance of HS and CS for proper activation and movement of signaling molecules. Surprisingly, sfl appears to be more disruptive than oxt on signaling. Ongoing experiments examine the role of the core proteoglycan Dally-like and look for signals that might upregulate cartilage deposition. These results will help to identify targets for the development of novel OA therapeutics. Supported in part by an HHMI undergraduate research award.

Abstract Number: 6312


Robert Sheehan

The Effects of Histone Modifications on Lens Cell Denucleation

Robert Patrick Sheehan and Melinda K. Duncan
Department of Biological Sciences, University of Delaware, Newark, DE, 19716

The lens of the eye contains both epithelial and fiber cells, with fibers cells deriving from the equatorial epithelium. When these cells differentiate, the nucleus and cellular organelles are broken down to facilitate transparency. However, small fragments of DNA can remain in fully mature lens fiber cells although its structure is unknown. We found that histone H3, trimethylated on Lysine 9, which is associated with compact heterochromatin, co-localizes with these DNA fragments. In contrast, the DNA remnants are not associated with either histone H3 Lysine 9 acetylation or histone H4 lysine 8 acetylation which is predominant in the more accessible euchromatin. These data suggest that the DNA fragments persisting in mature lens fiber cells are entirely composed of heterochromatin, perhaps because their highly compacted state prevented access of the nucleases responsible for DNA degradation during lens fiber cell differentiation. Supported by Howard Hughes Medical Institute and the National Eye Institute.

Abstract Number: 6387




Katharine Shelly

Localization of human sperm PMCA4b which is required for normal motility in mouse sperm

Katharine E Shelly, Rolands Aravindan, and Patricia A Martin-DeLeon
Department of Biological Sciences

Progressive and hyperactivated motility in sperm are both dependent on Ca²⁺and ATP to effect fertilization, with hyperactivated motility utilizing more ATP than progressive motility. The ATP-driven enzyme Plasma Membrane Calcium/calmodulin-dependent ATPase isoform 4b (PMCA4b) is the principal calcium efflux pump in mouse sperm, and its absence results in the loss of hyperactivated motility and fertility. Thus, PMCA4b plays an important role in murine sperm function. Protein BLAST analysis illustrates 84% identity and 91% similarity between the mouse and human PMCA4b sequences. Analysis of the functional domains suggests conservation of PMCA4b’s activity in human sperm. The objective of this investigation is to characterize the presence of PMCA4b in human sperm by Western blotting, as well as to determine its subcellular localization by immunocytochemistry (ICC). Flow cytometric analysis serves to confirm the presence of PMCA4b on the membrane. ICC studies should elucidate whether or not localization of human PMCA4b is present on the principal piece of the flagellum, which would coincide with findings in the mouse and suggest its involvement in human sperm motility. This work was supported by the Charles Peter White Fellowship and the NIH-COBRE grant #5P20RR015588-07.

Abstract Number: 6308

Laura Sloofman

Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition.

Mathematical modeling of elasticity changes in the cortical bone
of HIP/RPL29- deficient mice

Laura Sloofman, John A. Pelesko², Gilberto Schleiniger², and Catherine B. Kirn-Safran¹Departments of ¹Biological Sciences and ²Mathematics

Mathematical modeling, or using equations to describe a system, is a powerful technique in transgenic research. Our laboratory created the first viable mutant mouse model lacking an individual ribosomal protein (HIP/RPL29). A short stature phenotype associated with increased bone fragility is observed in HIP/RPL29 null mice. We recently noted that a decrease in collagen cross-linking during the growth of HIP/RPL29 null bone precedes an overall enhancement in the mineral-to-matrix ratio in adult bone. We hypothesize that we can build on existing mathematical models to describe the changes in bone structure and composition resulting from HIP/RPL29 deficiency. Hierarchical multi-scale models of wild type and HIP/RPL29 null cortical bone will be used to simulate adult control and mutant bone. We aim to relate the changes in null bone samples to elasticity measurements collected via three-point bending tests. Since the equations describing mutant and control bone are interrelated, we can extrapolate relationships among the mentioned properties. We will also use this model to predict the elasticity in control and mutant bones at three months of age. All calculations will be compared with accepted experimental values. This model will be altered as the three-month mutant bone phenotype is refined. Funding was provided by the Howard Hughes Medical Institute to LGS and NIH P20-RR016458 to CBKS.

Abstract Number 5191



Jamie Stull

Lenses from Connexin50 Mutant Mice Exhibit
the Unfolded Protein Response

Jaime K. Stull, Zeynep Firtina, and Melinda K. Duncan
Department of Biological Sciences

Cataract is the leading cause of blindness worldwide. Although the majority of cataracts are age-related, they may also be caused by heritable mutations. In humans as well as mice, congenital cataract occurs in Connexin50 (Cx50) mutants although the cataract phenotypes are more severe than that observed in Cx50 null mice. We hypothesized that when cells attempt to make Cx50 from a mutated gene, the resulting protein does not fold properly, inducing a cell-stress mechanism called the Unfolded Protein Response, UPR. We investigated this in mice harboring two different Cx50 mutations, Cx50^S50P/S50P and Cx50^G22R/G22R, that both exhibit similar lens phenotypes including microphthalmia, unorganized fiber cell structure, and cataract. We found a noticeable upregulation of the expression of the molecular folding chaperone BiP in mutant lenses after E15.5 in addition to an upregulation of XBP-1 gene expression and unconventional splicing indicative of the activation of the UPR sensor IRE1. These data further support our prior work suggesting that UPR can contribute to lens phenotypes due to mutations in genes whose products must transit the secretory pathway. Supported in part by an HHMI undergraduate research award.

Amy Styer

Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition.

Recipient of an ASBMB Undergraduate Travel Award.

Expression, Purification, and Characterization
of a New Sulfhydryl Oxidase from Trypanosoma brucei

Amy Styer, Vamsi Kodali, Vidyadhar Daithankar, and Colin Thorpe
Department of Chemistry and Biochemistry

A sulfhydryl oxidase from Trypanosoma brucei was expressed, purified and studied enzymologically for the first time. The protein is homologous to ALR (augmenter of liver regeneration), an essential flavin-linked enzyme which catalyzes disulfide bond formation in the mitochondrial intermembrane space (IMS) of eukaryotes. Trypanosomes lack a gene for Mia40, a necessary redox partner with ALR in yeast and mammals. Future research will determine how trypanosomes compensate for the lack of Mia40, and if this sulfhydryl oxidase has the same biological localization and function as ALR. We have shown that six of seven cysteines in the 33 kDa trypanosomal ALR are present as disulfide bonds. In oxygen electrode assays, the enzyme catalyzed disulfide-bond formation in the model substrate dithiothreitol (DTT), but not in the monothiols glutathione and cysteine. Molecular oxygen was a much better terminal electron acceptor for trypanosome ALR than for human ALR (TbALR Km = 15±1μM; some 15-fold lower than the human enzyme). Further studies are aimed at characterizing the kinetic mechanism of this flavoenzyme by stopped flow spectrophotometry. Understanding trypanosome redox biochemistry is important because Trypanosoma brucei, the causative agent of African Sleeping sickness, kills nearly fifty thousand people annually and current treatments are expensive and incur serious side reactions. This exploration of trypanosome mitochondrial IMS disulfide-bond formation may lead to biomedical advances in the fight against trypanosome diseases.
Funded by a Beckman Scholars Fellowship to A.S. and NIH GM26643 to C.T.

Laura Sloofman displays her Honorable Mention Award from the ASBMB Undergraduate Poster Competition in the Systems Biology Category.	Amy Styer displays her Honorable Mention Award from the ASBMB Undergraduate Poster Competition in the Protein Structure and Function Category.	Dr. White wonders how he will be able to pay for all of the big appetites.
Our Brief excursion into Disneyland.	UD Students enjoying pizza after the ASBMB Undergraduate Poster Competition	Judge Michael Cox (UD BA Biology '74) at Tejal's poster.
Amy, Michael, Rebecca, Seung, and Gary	Tejal, Megan, Jean, Kate, and Rachel	Tyler, Seung, and Amy
Yes, there really are palm trees in Southern California	Photos by David Usher and Hal White	Laura And Megan

Link to UDaily article.

The trip to the Experimental Biology 2010 Meetings in Anaheim was organized by the University of Delaware HHMI Undergraduate Science Education Program with additional support from travel grants from the American Society for Biochemistry and Molecular Biology, Arnold and Mabel Beckman Scholars Program, and the Women Scholars Program. The HHMI Undergradaute Science Education Program, the Arnold and Mabel Beckman Scholars Program, Charles Peter White Fund, Undergraduate Research Program, NIH, NSF, National Eye Institute, and DOD supported research by the students.

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Created 20 January 2010, last revised 11 May 2010 by Hal White [halwhite at udel.edu]
Copyright 2010 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware