Boston Convention Center

EB2013
EXPERIMENTAL BIOLOGY MEETINGS
Boston, MA  
APRIL 20-24, 2013




Boston Night Skyline

For the past 13 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 annual conference, the American Society for Biochemistry and Molecular Biology (ASBMB) sponsors its 17th Undergraduate Poster Competition in which 6 UD students participated this year. Since 2001, students from the University have received more awards in this competition than students from any other college or university.  This year, two of the six students entered in the ASBMB competition received honorable mention awards.

The University of Delaware group included four faculty and 9 undergraduates.



From left to right: Allison McCague, Christine Dang, Megan Dumas, Monica Pirigyi,
Helen Schmidt, Laura Powell, Zachary March, and Nicholas Lombardi (Kahina Ghanem not shown)

Prof. Hal White, Chem &Biochem
Prof. Dave Usher, Biol. Sci.
Prof. Seung Hong, Biol Sci
Prof. Gary Laverty, Biol Sci

Christine Dang (Salil Lachke)
Megan Dumas (Jia Song)
Kahina Ghanem (Gary Laverty)
Nicholas Lombardi (Erica Selva)
Zachary March (David Colby)
Allison McCague (Erica Selva)
Monica Pirigyi (Neal Zondlo)
Laura Powell (Fidelma Boyd)
Helen Schmidt (Eric Wommack)




University of Delaware students and their abstracts.

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<>Christine Dang
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Characterization of small Maf regulators in lens fiber cell differentiation and cataract formation
Christine Dang, Smriti A Agrawal, Stephanie M Waters, Hozumi Motohashi, Salil A. Lachke

The MAF (musculoaponeurotic fibrosarcoma) gene family includes “large” and “small”  MAF subgroups that encode bZIP transcriptional regulators. Although mutations in the large  MAF gene “MAF” cause human congenital cataract, function of the small MAF genes MAFF, MAFK, and MAFG in the lens remains uncharacterized.  iSyTE (integrated Systems Tool for Eye gene discovery), a tool that identifies genes associated with lens development and cataract, has predicted  MAFG as a potential regulator in the lens. To test this prediction, we aimed to characterize mouse models carrying targeted mutant alleles of small Maf genes.  In situ hybridization and Western blotting confirmed that  Mafg mRNA and protein were expressed in the embryonic and adult mouse lens tissue. Furthermore,  Mafg-/- ;Mafk+/- compound mutant mice exhibit lens defects and develop pre-senile cataract by age six months. Expression profiling by mouse Illumina microarrays on 2-month old  Mafg-/-;Mafk+/- compound mutant and control lenses identified several differentially regulated genes in the lens. This analysis indicates that expression of the heat shock protein gene Hspb1 (also known as  Hsp27) is significantly down-regulated in  Mafg-/-;Mafk+/- compound mutant lenses. Together, these results suggest a functional role for the small Maf proteins MAFG and MAFK in maintenance of lens transparency. This research was funded by a Fight For Sight Foundation grant



Megan Dumas
Recipient of an ASBMB Undergraduate Competitive Travel Award
 
The role of the small GTPase Arf6 and its potential regulation by miRNA-31 during early embryogenesis
Megan Dumas, Nadezda Stepicheva, and Jia L. Song
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716
MicroRNAs are small, non-coding mRNAs that repress protein translation and/or mRNA degradation.  microRNA-31 is one of the most abundant miRNAs in the sea urchin embryo, and  it is essential for proper cell specification.  We bioinformatically identified Arf6 to be a potential target of microRNA-31.  Arf6 mediates membrane trafficking between the plasma membrane and endosomal compartments, including receptor endocytosis and actin rearrangement. The function of Arf6 has not been examined in early development, and we hypothesize that Arf6 is regulated by microRNA-31 and may direct cell movement and specification of mesenchymal cells.  Quantitative, real time PCR and Western blotting were used to characterize Arf6. Arf6 protein levels in microRNA-31 knockdown embryos are increased compared to the control, suggesting that Arf6 may be regulated by microRNA-31. We are currently testing the function of Arf6 with morpholino antisense oligonucleotides, constitutively active (Q67L) and dominant negative (T27N) Arf6 mutants.  The Arf6 knockdown phenotypes include thickened filopodia, exogastrulation and vegetal cell detachment, indicating that Arf6 is critical for proper early development. This research provides insight into the function of Arf6 during early embryogenesis, its regulation by miRNA-31, and the role of miRNAs during early development.  This work is funded by the University of Delaware Research Fund.

      
                                                                                                                                                                                                                  

Kahina Ghanem

 Basolateral Transport of HCO3- by Avian Renal Proximal Tubule Cells in Culture
Kahina Ghanem and Gary Laverty
Department of Biological Sciences, University of Delaware, Newark, DE 19716

Bicarbonate (HCOˉ) is an important component of acid/base regulation. Therefore, it is crucial to understand the mechanism of bicarbonate reabsorption in the kidney proximal tubule (PT). In mammals the reabsorption mechanism is well understood, but evidence suggests that birds may use a different mechanism. We hypothesize that in the avian PT HCO3- crosses the luminal membrane in ionic form, rather than as CO2, and that HCO3- ions then leave the basolateral side via an NBC1-like transporter, as in the mammalian PT. The experiments used primary cell cultures of chick PT and electrophysiological studies on these monolayers to measure currents (ISC) associated with ion transport. Monolayers were first stimulated with 1µM forskolin, which activated a chloride secretory current in the avian PT. This was followed by basolateral application of 100 µM DIDS, an inhibitor of NBC1. With bicarbonate in the bathing solution DIDS caused an increase in ISC of 6.25 + 1.565 µAmps/cm2 (n = 6 ), but only   2.30  +  0.58 µAmps/cm2 (n = 5) in the absence of bicarbonate. The increased ISC is consistent with inhibition of electrogenic transport of HCO3- ions via the NBC1 transporter, and the decreased effect of DIDS in the nominal absence of bicarbonate further supports this. Additional studies support both mRNA expression and NBC1 protein expression (western blotting) in chick PT cultures and native tissue. Experiments are underway to test for an alternative apical transporter.





 Nicholas Lombardi


Investigating the Role of Wntless isoforms in Wingless Signaling
Nicholas Lombardi  and Erica Selva
Department of Biological Sciences, University of Delaware, Newark, DE 19716

Wnts are secreted signaling molecules that have essential functions in development and adult homeostasis. This family of proteins as well as the wnt signal transduction pathway is conserved from humans to invertebrates, including Drosophila. Wingless (Wg) is the founding member of the Drosophila Wnt family. Wg secretion forms a morphogen gradient that elicits different signals in its short and long-range targets. Wntless (Wls) is a highly conserved multi-pass transmembrane protein that binds to Wg and escorts it through the secretion pathway. Wls exists in two isoforms within the cell designated Wls-A and Wls-B.  The goal of this research was to determine the relative abundance of the two Wls isoforms, and determine the difference in their functions. PCR analysis results showed considerably higher levels Wls-B in the Drosophila imaginal wing discs. Since, wlsB is predominate in the wing disc I explored the phenotypic consequences of overexpressing each isoform in the posterior compartment of the developing wing disc. Overexpression of Wls-A in wing discs caused an increase in the distribution of Wg within receiving cells and a corresponding expansion of Distalless, a long-range target of Wg signaling.  In addition, the overexpression of Wls-A showed a shift of Wg from the apical to basal surface of the wing disc. Conversely, overexpression of Wls-B showed a decrease in Wg expression in the posterior compartment and a shift of Wg toward to apical surface and away from the basal surface even more than is observed in wild type tissue. Taken together these results suggest that the cellular function of Wls A and B differ. The detailed molecular, cellular and developmental consequences of these differences will be examined in future experiments.






Zachary March
Recipient of an ASBMB Undergraduate Travel Award
Recipient of Honorable Mention Award in The ASBMB Undergraduate poster Competition

Detection of pathological tau conformers in cerebrospinal fluid
Zachary M. March1, Sharad Gupta1,2, and David W. Colby1
1Department of Chemical and Biomolecular Engineering, University of Delaware
2Department of Biological Engineering, Indian Institute of Technology-Gandhinagar

Misfolded tau is a pathological hallmark of over 20 phenotypically distinct neurodegenerative disorders, including Alzheimer's disease (AD). Researchers are beginning to speculate that misfolded tau may spread throughout the brain from a single locus like a prion; whether ornot tau aggregates cross the cell membrane remains unclear. To investigate the presence of misfolded tau in cerebrospinal fluid (CSF)  of patients with AD, we developed an amyloid seeding assay for tau. Our findings indicate that misfolded tau molecules migrate in the CSF through the extracellular space, supporting the possibility that misfolded tau may spread through the brain like a prion. ZM supported in part by an HHMI undergraduate research
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Allison McCague
Recipient of Honorable Mention Award in The ASBMB Undergraduate poster Competition

 
The role of N-linked glycosylation during Drosophila development
Allison McCague and Erica M. Selva

Dept. of Biological Sciences, Univ. of Delaware, Newark, DE 19716

N-linked glycosylation is a key post-translational modification for many secretory pathway targeted proteins. Endoplasmic reticulum glycosyltranferases construct a 14-sugar precursor on a dolichol carrier, which is transferred to protein consensus sites. alg9 and alg10 N-glycosylation mutants were used to examine the role of this modification during Drosophila development. alg10 encodes the enzyme catalyzing terminal glucose addition to the sugar-precursor prior to transfer, while alg9 encodes an enzyme acting five steps earlier. In embryos, loss of alg9 and alg10 caused severe and pleotrophic defects. Central nervous system (CNS) neurons were specified in both alg9 and alg10 embryos. alg10 loss disrupted axon pathfinding, while alg9 embryos lacked mature neurons. Drosophila eye development in the absence of alg9 and alg10 yielded small rough adult eyes, but alg9 eyes were more severe. Examination of molecular markers in alg9 and alg10 late 3rd instar eye imaginal discs suggested adult small rough adult eyes might be due to neuronal apoptosis. These eye discs also showed defects in axon pathfinding, as shown by the loss of Bolwig’s nerve and disrupted axon tracks. These results suggest loss of alg10 may disrupt the maturation of a subset of N-glycoproteins, since alg9, which acts earlier, has more severe developmental defects. Funding sources: UD Undergrad Research Program, NSF and HHMI



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< style="font-weight: bold;">Monica Pirigyi
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Synthesis of Functional Proteins via Bioconjugation

Monica Pirigyi and Neal Zondlo
Department of Chemistry and Biochemistry, University of Delaware

Large protein synthesis is a present day biochemical challenge. Proteins can efficiently be expressed from various sources; however, post-translational modifications to introduce specific phosphorylation and spectroscopic labels can be challenging. One promising mechanism in creating these large, functional proteins is bioconjugation. This work is directed toward microtubule binding studies to elucidate the mechanism of tau aggregation in Alzheimer’s Disease. Peptides from the tubulin-binding domain of Tau were synthesized on an automated peptide synthesizer and modifications were conducted, on both solid and solution phase, to allow incorporation of specific phosphorylation events and multiple bioorthogonal functionalities. Peptides were modified and peptideconjugation reactions were conducted. To elucidate the efficiency of these bioconjugation reactions we have used model peptides and demonstrated clean, efficient conversions in aqueous solutions. Further, we are utilizing these tools to synthesize proteins with multiple tubulin-binding domain repeats and to study their ability to bind, polymerize, and depolymerize microtubules in both their nonphosphorylated and phosphorylated states.







Laura Powell

Regulation of the Osmotic Tolerance Response in the Halophile Vibrio parahaemolyticus
Laura Powell and Fidelma Boyd

Dept. of Biological Sciences, Univ. of Delaware, Newark, DE 19716

Vibrio parahaemolyticus is a Gram-negative, rod-shaped halophile bacterium present in marine environments worldwide.  The bacterium has an absolute requirement for NaCl and can grow in up to 10% NaCl in complex media.  Vibrio parahaemolyticus is also a pathogen of humans that causes gastroenteritis usually through the consumption of raw oysters.  The V. parahaemolyticus genome contains a large number of compatible solute synthesis and transporter systems that allows it to grow in high NaCl concentrations.  How these multiple systems and osmotic tolerance in general are regulated is unknown.  In this study, we investigated the regulation of osmotic tolerance by examining the homologue of the EnvZ/OmpR two-component signal transduction system, vp0155 and vp0154.  In Escherichia coli EnvZ/OmpR regulates more than 100 genes, and significantly influences cell growth, metabolism, and motility.  For example, in response to osmotic stress the EnvZ/OmpR system has been shown to regulate expression of outer membrane proteins (OMPs).  We examine its role in V. parahaemolyticus by deleting a portion of vp0155, an envZ homologue and vp0154, the ompR homologue.  Through splicing by overlap extension (SOE) PCR and homologous recombination, a deletion was created in both envZ and ompR.  Through homologous recombination and selection, mutants harboring in-frame deletions in the envZ and ompR genes were created, and confirmed by PCR and sequencing.  We describe the effects of our knockout mutations in envZ/ompR in response to NaCl, acid, and alkaline stresses as well as swimming and swarming motility and OMPs and type VI secretion system expression. Research supported by a grant from the Howard Hughes Medical Institute.


      






Helen Schmidt
Recipient of an ASBMB Undergraduate Travel Award

DNA polymerase phylogeny recapitulates virioplankton biology
Helen F Schmidt1, Eric G Sakowski1, Shannon J Williamson2, Shawn W Polson1, K Eric Wommack1
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University of Delaware, Newark, DE, 2Lake Pend Oreille Waterkeeper, Sandpoint, ID

Despite decades of investigation, we know little of the predominant biological characteristics of aquatic viruses that drive the rapid turnover of abundant virioplankton assemblages. DNA polymerases are essential to replication and thus enable connections between diversity and an important biological feature of viruses. Novel DNA polymerases, containing all critical functional domains, were common within dsDNA and ssDNA virioplankton metagenomic libraries. Finding DNA polymerases in ssDNA libraries was unexpected, as these genes have not been observed within ssDNA viruses. Comparison of PCR-amplified environmental DNA pol sequences with virioplankton metagenome sequences showed that the majority of the metagenomic polymerase sequences grouped away from previously identified groups of viral DNA polymerases forming a new group that included siphoviruses. An F762L amino acid substitution was observed within this dominant clade. Phylogenetic analysis of phages with known life cycles indicates that an F/Y762L substitution is a strong predictor of lysogeny, indicating that slower-replicating, lytic or lysogenic phage populations may predominate within the virioplankton and shape the influence of viruses on marine biogeochemical cycles. This work was funded by the HHMI Undergraduate Science Education program the NSF Microbial Genome Sequencing Program, the Gordon & Betty Moore Foundation, and Delaware EPSCoR.




Boston Daytime Skyline

Dinner at Durgin Park

Dinner at Durgin Park.
UD Alum Courtney Ngai at left.


UD ASBMB-Undergraduate Affiliate's
Activity Poster for 2012-2013.


Thousands of posters on the
Boston Convention Center main floor,


Welcome sign outside the Boston Convention Center
two days after the end of the Marathon Bommer capture.





The trip to the Experimental Biology 2013 Meetings in Boston 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. The HHMI Undergradaute Science Education Program, Charles Peter White Fund, Undergraduate Research Program, NIH, NSF, supported research by individual students.


Links to previous EB Meetings:
2001 in Orlando
2002 in New Orleans 2003 in San Diego
2004 in Boston
2005 in San Diego 2006 in San Francisco
2007 in Washington, DC 2008 in San Diego 2009 in New Orleans 2010 in Anaheim 2011 in Washington, DC
2012 in San Diego

Return to  University of Delaware HHMI Home Page
Created 7 January 2013,  Last revised 29 April 2013 by Hal White [halwhite at udel.edu]
Copyright  2013 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware