San Diego, CA   APRIL 1-6, 2016

For the past 17 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 (ASBMB) sponsored its 20th Undergraduate Poster Competition in which 8 UD students participated. Since 2001, students from the University have received more awards in this competition than students from any other college or university.  UDaily Article.

The University of Delaware group included three faculty and 8 undergraduates.

Students from left to right; Gabriel Gregorzak, Hannah Wastyk, Tyler Heiss, Nicole Wenzell, Josh barton, Morgan Thomas, Jay Subramoney, Blair Schneider
Prof. Hal White, Chem & Biochem
Prof. Seung Hong, Biol Sci
Prof. Gary Laverty, Biol Sci

Josh Barton, Biology
Gabe Gregorzak, Chemistry
Tyler Heiss, Chem and Biochem
Blair Schneider, Biology
Jay Subramoney, Chem and Biochem
Morgan Thomas, Biology
Hannah Wastyk, Chem and Biochem
Nicole Wenzell, Chem and Biochem

University of Delaware students and their abstracts.

Josh Barton

Molecular Characterization of Two Human Lens Epithelial Cell Lines and Their Suitability to Study Function of Cataract Genes

Joshua Barton, Archana D. Siddam, Deepti Anand, Salil A. Lachke.

Biological Sciences, University of Delaware, Newark, DE

The ocular lens is a transparent tissue that focuses light on the retina, allowing high-resolution vision. The loss of lens transparency causes an eye defect termed cataract, which is the leading cause of blindness worldwide. Half of the U.S. population above age 80 is affected by cataracts, for which surgical treatment is the principal therapeutic intervention. In 2014, over 3.6 million cataract surgeries were performed in the United States, and over 20 million surgeries were performed globally. Age-related cataracts are caused by a variety of stress conditions including elevated oxidative stress, increased sugar (diabetes), smoking or environmental insults such as exposure to ultraviolet (UV) radiation. Thus, characterization of biological and environmental factors that affect lens transparency is the first critical step toward the development of new therapies for cataracts. Recent discoveries have revealed a surprising link between proteins that are components of cytoplasmic RNA granules (RGs) and mammalian cataract. RGs like Processing Bodies (PBs) and Stress Granules (SGs) represent specialized cytoplasmic sites for regulation of RNA in the control of gene expression. PBs, the constitutive class of RGs, are RNA-protein complexes that silence or channel mRNA to decay, and therefore function to regulate the cellular proteome. SGs form as an early reaction to cellular stress, specifically allowing the translation of proteins that function in homeostasis. To investigate RG function in lens cells, we molecularly characterized two human lens epithelial cell lines (LECs), SRA01/04 and HLE-B3. These previously uncharacterized cell lines can serve as valuable in vitro tools for investigating the intricate molecular basis for human cataractogenesis. It is important in cell culture studies to validate the origin and identity of the cell line. Therefore, our first step was to confirm that the cell lines were of human origin. Analyzing genome-specific Short Tandem Repeats by PCR, we authenticated that the LEC lines are indeed human-derived. We next analyzed gene expression in these LECs by high throughput Illumina microarray analysis to investigate the extent of their retention of lens-like character. The microarray data demonstrates that both LECs retain expression of genes that are expressed and/or enriched in normal lens epithelial cells. Using a bioinformatics tool “iSyTE” developed to predict genes implicated in lens biology and cataract, we further validated the expression of cataract associated genes in these LECs using RT-PCR. Furthermore, we find that both LECs support formation of RGs, and exhibit formation of SGs under various stress conditions such as chemical and UV-stress. Thus, these studies present the first cellular models for investigating the molecular biology of UV-radiation exposure in lens epithelial cells, and therefore represent a new resource for study of factors that are linked to age-associated cataract in humans.
Support or Funding Information

Funding was provided by the 2015 Delaware Governor's Bioscience Fellowship to Joshua Barton, and University of Delaware Startup funds as well as a grant from The Pew Charitable Trusts awarded to Salil Lachke.

Abstract No. 1058.1      Poster No. C66    Tuesday,  April 5

Gabe Gregorzak
Synthesis of Artificial Bacterial Cell Wall Fragments as Tools to Study the Innate Immune System

Gabriel Gregorzak, James Melnyk, Kristen DeMeester, Catherine Leimkuhler Grimes

Department of Chemistry & Biochemistry, University of Delaware, Newark, DE

During a bacterial infection, the body’s first line of defense is provided by the innate immune system. This system is triggered by pathogen-associated molecular patterns (PAMPs) that are recognized by different members of the body’s innate immune system called pattern recognition receptors (PRRs). One such PRR of interest is nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mammalian Nod2 is a cytosolic protein that binds to and signals in response to carbohydrate containing bacterial cell wall fragments; the smallest of which is muramyl dipeptide (MDP). Mutations in Nod2 increase the likelihood of the development of Crohn’s Disease and other chronic inflammatory diseases. Previously, our lab has shown that Nod2 binds directly to MDP, but currently the molecular mechanisms of activation and binding are still unknown1. Currently, we are addressing this issue using synthetic chemistry. This project focuses on the synthesis of an assortment of MDP dimers, which vary in the length and functionality of their connecting linkers. To avoid troublesome late-stage de-protection issues, the synthesis diverges in its early stages to result in two differing MDP-like fragments. These two fragments can then be coupled together and de-protected, resulting in the MDP dimer.  Once synthesized, these dimers will be used in in vitro SPR binding assays, as well as in cell-based assays to elucidate the binding and activation mechanisms used by Nod2. Ultimately, these synthetic tools will aid in the advancement of our understanding of how are body’s sense and respond to bacteria.
GG Supported Howard Hughes Medical Institute Undergraduate Summer Scholars award

Abstract No. 612.4      Poster No. C202      Sunday, April 3

Tyler Heiss

Chemical and Chemoenzymatic Synthesis of UDP-N-Acetyl Glucosamine Probes

Tyler Heiss, Kristen E. DeMeester, Hai Liang, Borja Barbero, and Catherine Leimkuhler Grimes

Department of Chemistry and Biochemistry, University of Delaware, 163 The Green, Newark, DE, 19716

Uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is a ubiquitously used nucleotide sugar involved in bacterial cell wall biosynthesis and the posttranslational modification GlcNAcylation. If functionalized versions of UDP-GlcNAc were more easily accessible, these chemical probes could be used to rapidly investigate biological systems. However, current chemical strategies for synthesizing UDP-GlcNAc derivatives are difficult and lengthy. Herein we describe the chemoenzymatic synthesis of strategically functionalized UDP-GlcNAc chemical probes from Glucosamine-1-Phosphate (GlcN-1-P) and synthetic acetyl donors known as N-acetylcysteamine thioesters (SNAcs) using the bacterial cell wall biosynthesis enzyme GlmU. Hypothesis: If GlmU is shown to use GlcN-1-P and derivatized SNAcs to produce modified UDP-GlcNAc’s, then these altered building blocks will be used to investigate bacterial cell wall biosynthesis and O-GlcNAc transferase (OGT) activity.

Acknowledgments and Funding: We would like to thank the Koh Lab and the rest of the Grimes Group for their continuing help and support. We also thank the Howard Hughes Medical Institue (HHMI), the University of Delaware’s Summer Scholars program, and the Pew Foundation for funding my work.

Recipient of an ASBMB Undergraduate Travel Award
Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition.
Abstract No.  612.3    Poster No. C201     Sunday, April 3

Blair Schneider


Blair Schneider1, Colin Kern2, Allen Hubbard 3, Wayne Treible 4, John W. Finger Jr. 5 , Tracey Tuberville 6 ,
Travis C. Glenn 5 , Matt Hamilton 6 , Susan J. Lamont7, Carl J. Schmidt8

1Department of Biological Science, University of Delaware, Newark; 2Department of Animal Science, University of California, Davis; 3Department of Bioinformatics and Systems Biology, University of Delaware, Newark; 4Department of Computer and Information Science, University of Delaware,  Newark; 5Department of Environmental Health Science and Interdisciplinary Toxicology Program, University of Georgia, Athens; 6Savannah River Ecology Laboratory, University of Georgia,  Aiken, 7Department of Animal Science, Iowa State University, Ames; 8Department of Animal and Food Sciences, University of Delaware, Newark,

            The domestic chicken is very important in its use as a model organism for understanding regulation of numerous biological processes in avians. To better understand energy storage and mobilization, this work focuses on defining the transcriptome of chicken abdominal and cardiac adipose tissue.  Transcriptome libraries were prepared from Illinois abdominal and cardiac adipose samples with the following procedure: 1. Isolation of mRNA using mirVana™ miRNA Kit, 2. Verification of mRNA quality and integrity by Fragment Analysis, 3. Transcriptome libraries prepared using Illumina Stranded RNA Prep Kit and sequenced at the Delaware Biotechnology Institute Core Sequencing Facility, 4. Transcription levels determined using the  fRNAkenseq software, and 5. Identification of differentially expressed genes, data analysis, functional clustering and pathway analysis.  To begin our analysis we identified genes that were differentially expressed between the abdominal and cardiac fat tissue.  This identified differentially expressed transcripts encoding adipokines, transcription factors and enzymes involved in processing fat.  The same procedure was performed on alligators, and the gene expression was analyzed in their fat bodies.  Current effort focuses on comparing alligator gene expression with that obtained from chicken adipose tissue to better understand the evolution of this tissue in Archosaurs.


Acknowledgements: This material is based upon work supported by the National Science Foundation under Grant No. 1147029 and by the Agriculture and Food Research Initiative Competitive Grant No. by NIFA 2010-04233 from the USDA National Institute of Food and Agriculture.

Abstract No. 654.2    Poster No. C382      Sunday, April 3

Jay Subramoney

Chemical Ligation Synthesis of Isotope-Labeling Compatible Selenoprotein S

Jay Subramoney, Zhengqi Zhang, Rujin Cheng, Sharon Rozovsky

University of Delaware Department of Chemistry and Biochemistry, Newark, DE 19706 

 Selenoproteins contain the 21st amino acid selenocysteine (Sec) and play vital roles in human health, such as antioxidant defense. Although selenoproteins are expressed naturally in the human body, procuring sufficient quantities for biochemical and biophysical characterization by heterologous expression is challenging.  Low yield in genetic incorporation is due to the Sec codon being identical to the UGA stop codon in mRNA. An alternative approach for their preparation is chemical ligation, a semi-synthetic method that relies upon an amide bond formation to generate peptides and proteins from their complementary fragments. Here, we describe a new method for chemical ligation of selenoprotein S (SelS) which is compatible with simple and low cost isotopic labeling and does not rely on complete chemical synthesis of a selenoprotein. SelS is a member of the endoplasmic reticulum associated protein degradation (ERAD) pathway, a protein machinery devoted to relocating misfolded proteins from the ER to a cytoplasmic protein degradation complex. The active site lies close to the C-terminal where a sole Sec is paired with a Cysteine 13 residues away. Previously we have characterized the activity of SelS prepared by heterologous expression, however, structural characterization with the Sec in place was prohibited by the low yield. In our approach a Sec-containing fragment is expressed in E. coli and used as a source of the selenopeptide during the chemical ligation. This has the advantage of a high yield, with simple and efficient ligation while being compatible with isotopic-labeling of selenoproteins.  Useful applications of this method include incorporation of Selenium-77 and Carbon-13 for structural and biochemical characterization by NMR spectroscopy.

Abstract No. 595.5      Poster C83       Sunday, April 3.

Morgan Thomas

The Role of N-linked Glycosylation in Drosophila Development

Morgan Thomas and Erica M. Selva

Department of Biological Sciences, University of Delaware, Newark, DE

Asparagine-linked or N-linked glycosylation is an important post-translational modification pathway that adds 14-sugar oligosaccharide chains to the Asparagine of specific Asn-Xaa-Ser/Thr sequences in target proteins on the luminal side of the endoplasmic reticulum. These glycan “tags” are necessary for multiple cell functions including cell-cell recognition, extracellular signal regulation, and proper protein folding. The focus of this study is to examine the effect of loss of function mutations in the N-linked pathway during development. A group of human diseases, congenital disorders of glycosylation (CDG), arise from mutations in the genes involved in various steps of this pathway. CDGs display severe and pleotropic phenotypes, which reflect the universal importance of this protein modification, but most cases display phenotypes that arise from impaired neuronal function. The two specific genes under study in this project are alg9 and alg10. Each gene encodes a glycosyltranferase that adds a sugar residue to the growing oligosaccharide chain before it is transferred en masse to a target protein. The Drosophila eye is used as a model organ to study the effects of loss-of-function mutations in these genes as it serves as a surrogate for neuronal development. In adult flies, these mutations yield a small rough eye phenotype, which is more severe in alg9, as it acts five steps before alg10 in the pathway. In order to determine the molecular basis of this phenotype, larval eye discs that are homozygous mutate for alg9 and alg10 were dissected, stained for different glycoprotein and neuronal markers, and then imaged using confocal microscopy. Using ELAV, a neuron specific marker, we observed normally differentiated neurons in the eye disc.  However, these mutations interrupt proper glycoprotein trafficking, as Chaoptin, a photoreceptor specific cell surface adhesion molecule, accumulates in the cell bodies of alg9 and alg10 mutant photoreceptors. Presence of cleaved Caspase-3 later in eye development suggests that the accumulation of glycoprotein in the cell body eventually leads to photoreceptor apoptosis. Photoreceptor death likely continues through pupal development resulting in the reduced number of photoreceptors seen in alg10 adult eyes and an almost complete absence in alg9 adult eyes. These results indicate that CDG patients may have normal neuronal specification and differentiation, but experience neuronal deficits due to intracellular accumulation of glycoproteins leading to cell death. These data also suggest induction of the unfolded protein response (UPR) resulting in apoptosis may play a role in this process. Markers of endoplasmic reticulum stress and the unfolded protein response such as XBP1 and BiP will be studied in the future.
Support or Funding Information

Howard Hughes Medical Institute Undergraduate Summer Fellowship, University of Delaware Supply and Expense Grants
Abstract No. 619.1     Poster No. C236       Sunday , April 3
Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition.

Hannah Wastyk

Biochemical Characterization of the Interaction between Innate Immune Receptor Nod2 and its Chaperones

Hannah C. Wastyk1, Vishnu Mohanan2, Amy Schaefer1, Ching-Wen Hou1, Mackenzie Lauro1, Catherine Leimkuhler Grimes1,2

1Chemistry and Biochemistry, 2Biological Sciences, University of Delaware, Newark, DE, 19716

The human body houses over 10 trillion bacterial cells, of which our innate immune system must delineate between pathogenic and commensal. Disease can arise during misregulation of this bacteria, and one example is the debilitating inflammatory bowel disorder, Crohn’s disease (CD). The molecular pathology of CD is known to be a mutation in Nod2, an innate immune receptor responsible for binding directly to bacterial cell wall fragment, muramyl dipeptide (MDP). MDP has recently been shown to stabilize Nod2 in a cell based assay, and after binding to MDP, Nod2 signaling proteins activate the NF-κB pathway, a protective inflammatory response. In Crohn’s variants, however, Nod2 is rapidly degraded, thereby the downstream signaling is disrupted. Our initial work identified Hsp70 as a chaperone molecule that binds to and stabilizes Nod2. The purpose of this study is to characterize this interaction and stabilization effect, such that the natural stabilization of Nod2 within the cell can be extrapolated to a possible treatment option that mimics Hsp70’s activity on unstable Nod2 Crohn’s variants. Hsp70 is a highly ubiquitous protein that is able to confer selective stability by tuning its specific activity via a number of different cochaperones. Our data shows that Hsp40 contributes to the chaperone complex, and through limited proteolysis, we show that both Hsp70 and Hsp40 mediate Nod2 stability in vitro, and that this stability is further enhanced with the addition of Nod2’s ligand, MDP. Furthermore, we have biochemically characterized the importance of Hsp70’s ATPase activity for Nod2’s stability. Significance of this study extends beyond CD, as understanding Hsp70’s action and determining a method of control for Nod2 could be extrapolated to stabilize a large number of disease-relevant mutated proteins.
Acknowledgements: The authors would like to thank the following sources of funding: the Chemistry-Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through NUCLEUS.

Recipient of an ASBMB Undergraduate Travel Award
Recipient of a First Place Award in the ASBMB Undergraduate Poster Competition.
Abstract No. 811.2       Poster No. C84      Monday, April 4

Nicole Wenzell


Nicole A. Wenzell, Himal K. Ganguly, Anil K. Pandey, Glenn P.A. Yap and Neal J. Zondlo*

Department of Chemistry and Biochemistry, University of Delaware, 339 Brown Laboratory, Newark DE 19716

An nπ* interaction is a local interaction that orders two consecutive residues by delocalizing the lone pair of electrons from oxygen of the donor carbonyl to the antibonding orbital of the acceptor carbonyl carbon. This interaction is a recent discovery that gives insight into the inherent biases that proteins have to drive them to their folded state. By changing the electronics of the donor carbonyl on model proline-based peptides, we can tune the strength of this nπ* interaction. Proline can readily populate both the trans and cis conformations in a peptide bond. The nπ* interaction is shown to stabilize the trans conformation. Thus, in our model system the equilibrium constant of trans-cis isomerization (Ktrans/cis) acts as a readout for the strength of the nπ* interaction. Greater electron donation on the donor carbonyl causes a stronger interaction, resulting in higher Ktrans/cis values. The electronics of the donor carbonyl in the model peptides were modulated by synthesizing a series of derivatives with varying electron-donating capabilities on the donor carbonyl. The series of derivatives synthesized were selected in order to compare their electron donating capabilities to their nπ* interaction strength. The derivatives synthesized for analysis were, Boc, pivaloyl, formyl, acetyl, monochloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl. These successfully synthesized derivatives provide a range of electronic character to be analyzed for the strength of the nπ* interaction in each compound. The determination of the strength of the nπ* interaction was accomplished through combined analysis via 1H and 13C NMR and X-ray crystallography. NMR analysis provides the Ktrans/cis values for each derivative and thus the strength of the nπ* interaction. Electron donation involved in the nπ* interaction is seen via the chemical shifts of the 13C NMR. The chemical shift of the acceptor carbonyl moves downfield with stronger electron-donating substituents, while carbonyls uninvolved in the interaction remain constant. X-ray crystallography of the model peptides reveals insights into the nπ* interaction as it indicates the distance and angles of the interaction. Specifically, the more electron donating character of the acetyl compared to the formyl group results in a 0.3Å shorter distance (3.3-3.0Å) and an angle (115˚vs 90˚) that more closely resembles an ideal nucleophilic attack (109.5˚). The comparison of the Ktrans/cis values, the carbonyl chemical shifts, and the angles and distances of the donor-acceptor carbonyl pair serve as direct readouts for the strength of the nπ* interaction, which varies depending on the electron donating character of the donor carbonyl.

Recipient of an ASBMB Undergraduate Travel Award

Abstract No. 598.8      Poster No. C107       Sunday, April 3

Bonobo at the San Diego Zoo

San Diego from our plane as we departed.

Moma and baby orangutan
at San Diego Zoo

Flamingos at San Diego Zoo

Aerial Tram at San Diego Zoo

The Marriott and the San Diego
Convention Center from the air

The Marriott Hotel in the morning fog

Can you find your poster?

Dinner at Sovereign

Dinner at Sovereign

Snowy Mountains in New Mexico

Hannah receiving her award

Dinner at Chianti

The trip to the Experimental Biology 2016 Meetings in San Diego was organized by a legacy from the University of Delaware HHMI Undergraduate Science Education Program with additional support from travel grants from the American Society for Biochemistry and Molecular Biology.

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 2013 in Boston 2014 in San Diego
2015 in Boston

Return to  University of Delaware HHMI Home Page
Created 18 March 2016,  Last revised 15 April 2016 by Hal White [halwhite at udel.edu]
Copyright  2016 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware