OSU Life Sciences Interdisciplinary Graduate Programs Symposium 2016

Poster abstracts

1. Social Environment Modulates the Microglial Response to Ischemia Through Sex-Specific Mechanisms

Monica M. Gaudier-Diaz (Department of Neuroscience), Ning Zhang (Department of Neuroscience)

Abstract not available online - please check the printed booklet.

2. Zebrafish skeletal muscle as a surrogate system for studying human cardiomyopathy mutations: a progress report

Brit L. Martin (Physiology and Cell Biology, The Ohio State University), Tom L. Gallagher (Department of Molecular Genetics, The Ohio State University), Jonathan P. Davis (Physiology and Cell Biology, The Ohio State University), Brandon Biesiadecki (Physiology and Cell Biology, The Ohio State University), Christine E. Beattie (Department of Neuroscience, The Ohio State University), Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

3. Translation Control of the Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P

Andrei Rajkovic (Microbiology), Anne Witzky (Microbiology)

Abstract not available online - please check the printed booklet.

4. Identification of Replication-Dependent and Replication-Independent Linker Histone Complexes

Pei Zhang (Department of Biological Chemistry and Pharmacology, The Ohio State University), Owen E. Branson (Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University), Michael A. Freitas (Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University), Mark R. Parthun (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We identified proteins that bind to all histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability. These findings suggest that association with variant-specific binding partners may regulate linker histone dynamics.

Keywords: chromatin, linker histone, histone chaperone

5. Hybrid-Phase Ligation and Convergent Solid-Phase Ligation for Efficient Protein Total Synthesis

Ryan Yu (OSBP), Ziyong Hong (Chemistry)

Abstract:
Protein total synthesis is a powerful technique that allows the preparation of chemically diverse proteins not possible using conventional methods. Here, we develop hybrid solid-solution phase native chemical ligation that combines the efficiency of ligation on solid-phase with the yield of ligation in solution-phase. We demonstrate the effectiveness of this method through the synthesis of histone H4 and CENP-A proteins from four and five peptide segments.

Conventional convergent ligation approaches reach an effective size barrier through the chemical limits of solid phase peptide synthesis combined with practical limits of multiple solution ligation/purification steps. We demonstrate here preliminary results in convergent hybrid phase ligation, which has the potential to overcome these limits. Here, short peptide segments can be ligated on the solid phase to generate large ligation-compatible segments not accessible through direct synthesis. These large segments can be released from resin and ligated in solution to produce the full-length protein. We describe preliminary results in developing ligation handles compatible with this approach in the context of CENP-A and the 233-residue linker histone H1.2.

Keywords: peptide synthesis, post-translational modifications, histone

6. Clathrin mediated endocytosis is an indicator of membrane tension in spreading and migrating cells

Nathan M. Willy, Josh Ferguson (Physics)

Abstract:
Clathrin mediated endocytosis (CME) is a process by which receptor-bound macromolecules are internalized by pinching off areas of the plasma membrane. The dynamics of CME can be observed by tracking the formation and dissolution of clathrin coated structures via fluorescence imaging of tagged clathrin coat components. Since the process of CME involves deforming the plasma membrane, its dynamics are tied to the local membrane tension. We present data showing the relationship between clathrin dynamics and various naturally occurring cell behavior such as spreading onto a substrate and migration. It is our hope that observation of CME dynamics can be used as a proxy measurement for membrane tension with spatial and temporal specificity where direct measurement is invasive, cumbersome and limited to a single location.

Keywords: Clathrin, Plasma Membrane, Tension

7. Interactions between corticospinal and reticulospinal outputs determine muscle response in the upper limbs as revealed with stimulus-triggered averaging

Sarah Hulbert (Biophysics), Hojjat Adeli (Department of Civil Engineering and Geodetic Science, Department of Neuroscience), John Buford (Department of Physiology and Cell Biology, Division of Physical Therapy, Collge of Medicine, Center for Brain and Spianl Cord Injury Repair)

Abstract not available online - please check the printed booklet.

8. Title not available online - please see the printed booklet.

Matthew Plumb (MCDB), Jocelyn O. Eidahl, Nikoloz Shkriabai, Ross C. Larue, Jacques J. Kessl (College of Pharmacy, Center for Retrovirus Research, The Ohio State University), Mike Sweredoski, Sonja Hess (Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA), Zeger Debyser, Jan De Rijck (Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Leuven, Flanders, Belgium), Min Li, Robert Craigie (Laboratory of Molecular Biology, National Institute of Diabetes & Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD), Robert Gorelick (AIDS and Cancer Virus Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD)

Abstract not available online - please check the printed booklet.

9. Title not available online - please see the printed booklet.

Christopher Schwebach (MCDB), Elena Kudryashova (Chemistry and Biochemistry), Dmitri Kudryashov (Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

10. Title not available online - please see the printed booklet.

Xinyu Zhou (OSBP), Ki Ho Park (Department of Surgery, Davis Heart and Lung Research Institute, Wexner Medical Center, The Ohio State University), Pei-hui Lin (Department of Surgery, Davis Heart and Lung Research Institute, Wexner Medical Center, The Ohio State University), Hua Zhu (Department of Surgery, Davis Heart and Lung Research Institute, Wexner Medical Center, The Ohio State University), Hiroshi Takeshima (Department of Biological Chemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Japan), Jianjie Ma (Department of Surgery, Davis Heart and Lung Research Institute, Wexner Medical Center, The Ohio State University)

Abstract not available online - please check the printed booklet.

11. Title not available online - please see the printed booklet.

Sizhun Li (Molecular Genetics), Aaron N Bruns (OSBP), David M Bisaro (Molecular Genetics)

Abstract not available online - please check the printed booklet.

12. Associations among tissue vitamin D metabolites and breast cancer risk factors in women undergoing reduction mammoplasty

Renny S. Lan (Molecular, CEllular and Developmental Biology Program), Adana A. Llanos (Rutgers Cancer Institute of New Jersey, Rutgers University), Theodore M. Brasky, Catalin Marian (The Ohio State University Comprehensive Cancer Center), Ramona G. Dumitrescu (Saba University School of Medicine, Saba, Dutch Caribbean), Bhaskar V. S. Kallakury (Lombardi Comprehensive Cancer Center, Georgetown University), Jo L. Freudenheim (Department of Social and Preventive Medicine, University at Buffalo, State University of New York)

Abstract not available online - please check the printed booklet.

13. Title not available online - please see the printed booklet.

Brooke M Britton (Molecular Virology, Immunology, and Medical Genetics), Jiaquan Liu (Molecular Virology, Immunology, and Medical Genetics), Richard Fishel (Molecular Virology, Immunology, and Medical Genetics)

Abstract not available online - please check the printed booklet.

14. Title not available online - please see the printed booklet.

Grace Cooper-Olson (Ohio State Biochemistry Program), Jeff Kuret (Biological Chemistry and Pharmacology)

Abstract not available online - please check the printed booklet.

15. From Active to Silenced: Understanding How Repressing Chromatin Modification is Initiated at Transposons

Sarah Choudury (MCDB, Molecular Genetics Dept), Andrea McCue (Molecular Genetics Dept), Alissa Cullen (Molecular Genetics Dept)

Abstract not available online - please check the printed booklet.

16. Dielectric properties of biomolecules: Methods and Application to DNA oligomer

Mithila Agnihotri , Sherwin Singer

Abstract:
The frequency-dependent dielectric response of DNA in solution probes the dynamics of the
nucleic acid polymer and its counter-ion atmosphere on time scales important for cellular
processes. Recently, the dielectric spectra of short-chain DNA oligomers were measured,
exhibiting an absorption peak corresponding to a 10ns timescale process2. This is the first time
such data is available for oligomers small enough to be accessible to detailed molecular
dynamics simulations. Despite the theoretical and computational challenges described below,
we have been able to confirm that theoretical models can reproduce the 10ns experimental
feature. Our work is now focused on interpretation, and to resolve the conflicting interpretations
which have been proposed in the literature. The dielectric response of DNA is also important
because it can be harnessed to detect trace amounts of DNA as well as manipulate their
motions in micro- and nano-scale biomedical devices1.
The link between DNA dynamics on a molecular level and dielectric response has eluded
scientists for a long time. These properties are not revealed by experimental data alone.
Probing these properties using simulations has proved difficult as well due to large system size,
and long time-scales associated with their motions. Hence one needs to accumulate very long
trajectories, and for which the storage requirements are actually more severe than the
computational requirements needed to generate the trajectory. We have re-formulated the
theory of electrokinetic and dielectric processes in a way that enables calculation of dielectric
properties from both equilibrium and non-equilibrium simulations using sparse sampling of data
without compromising accuracy. We have shown proof-of-principle by calculating the electric
field-induced flow of a simple electrolyte system, and the contribution of ions to its dielectric
spectrum using our very sparse time sampling method. For DNA studies describe above, we
have accumulated an exceptionally long 5 microsecond trajectory for a DNA dodecamer
solvated with 11257 water molecules, and have been able to extract the dielectric spectrum
from sparsely stored data.

References:
1. R. Holzel, Dielectric and dielectrophoretic properties of DNA. IET Nanobiotechnol., 2009, Vol. 3,
Iss. 2, 28– 45
2. Omori S. et al. Dielectric dispersion for short double-strand DNA. Physical Review E, 2006, 73,
050901(R)

Keywords:

17. Consensus, Correlation and Combinatorics based approaches in engineering Triosephosphate Isomerase Stability

SIDHARTH MOHAN (Biophysics), NICHOLAS W. CALLAHAN (Biophysics), KIMBERLY R. STEPHANY (Department of Chemistry and Biochemistry, OSU), BRANDON J. SULLIVAN (Department of Chemistry and Biochemistry, OSU)

Abstract:
Nature exploits many protein folds, but we have only recently begun to understand the consequences of amino acid changes in a utilitarian manner. The conservation and co-variation of residues across homologous protein sequences throws light on the natural strategies employed in protein stability, and we can use statistical measures as a means to understand them. We have adapted relative entropy and mutual information metrics to reliably predict stabilizing mutations, and have begun to use combinatorial approaches to further our understanding of the meanings of amino acid consensus and correlations. Previously, a consensus variant of TIM (cTIM) suffered overall destabilization and impaired activity. However, a curated database of sequences yielded a variant with a native-like fold and activity (ccTIM). Since the variants differed minimally at the sequence level, we suspected the scrambling of statistical correlations in cTIM and developed combinatorial libraries to interrogate the exact nature of the differences between cTIM & ccTIM. Preliminary data points to global stabilization of the protein fold as a basis for the rescue of cTIM. We have developed a consensus-screening algorithm that has been applied to candidate TIM sequences from different branches of phylogeny in an attempt to exploit amino acid conservation & co-variation. Our results show a ~90% probability in identifying stabilizing mutations, with successful application to proteins with different folds. While we have begun to make rational mutations in ccTIM in an attempt to understand stability from the angle of destabilization of the fold, we hope to use positional binomial libraries and DNA-shuffling to further our knowledge of protein stability in this context.

Keywords: stability, engineering, library

18. Protein engineering approaches to understand the role of surface residues in the four helix bundle model protein Rop

Nishanthi Panneerselvam (Biophysics)

Abstract:
Deciphering the relationship between protein sequence and structural stability is important in understanding the effects of mutation on protein folding. This has applications in both industrial and therapeutic protein design.Rop is a 63 aminoacid residue four-helix bundle protein that controls the ColE1 plasmid copy number in E. coli. It has a well developed GFP assay for in vivo activity which helps us screen for best mutants after enrichment for active variants. Cells with active Rop tend to grow faster as the ones with inactive ROP have a higher metabolic load. Surface residues and surface electrostatics seem to play an active role in maintaining protein stability. A combinatorial library randomizing five surface positions in Rop to NNK (K=G or T) codons was constructed in a previous study. The surface library randomized five residues E39, S40, D43, D46 and E47. Interestingly, the consensus was positively charged residues in the place of negatively charged residues from wild-type. Specially, lysines were found more than arginines. To delve into this, all these five positions were individually mutated to Lys and Arg. The stability and activity data show some interesting differences between the Lys and Arg mutants, where one wouldn’t expect a surface point mutation to make a big difference. Free energy calculations are in progress using Gibbs-Helmholtz analysis. A related study is generating all possible point mutants in ROP. If all the possible point mutants in a protein are subject to moderate selection for activity or function, deep sequencing (high-throughput) can measure the relative abundance of mutants in a bulk competition experiment which in turn gives the relative fitness of each mutant. Preliminary data includes proof-of-principle selection experiments in three positions of Rop where variants were enriched 1,000 fold for activity. Analyzing the fitness landscape of Rop will yield each mutant’s competitive advantage/disadvantage.

Keywords: Protein engineering, Protein stability, Surface residues

19. Protein Dynamics underlies Cre-loxP DNA Recombination

Aparna Unnikrishnan (Biophysics Graduate Program), Carlos D. Amero (CIQ, Universidad Autnoma del Estado de Morelos, Mexico), Mark P. Foster (Department of Chemistry and Biochemistry, OSU)

Abstract not available online - please check the printed booklet.

20. Mechanistic Understanding of Fe-S Cluster Transport in X-linked Sideroblastic Anemia

Stephen A. Pearson (Ohio State Biophysics Program), Christine Wachmowsky (Ohio State Biochemistry Program)

Abstract:
Iron-sulfur (Fe-S) clusters are common cofactors found in essentially all living organisms. In humans, Fe-S clusters are made predominately in the mitochondria, but assembly machinery is also present in the cytosol [1]. However, when mitochondrial Fe-S cluster assembly is inhibited, some cytosolic Fe-S cluster proteins are not able to convert to their holo forms due to lack of cluster [2]. One hypothesis is that an Fe-S cluster precursor molecule is transported out of the mitochondria, but we believe this molecule to be the Fe-S cluster itself, coordinated to four glutathione molecules {[Fe₂S₂](GS)₄} [1,3]. Through liposome transport studies, we have shown that ABC transporter S. cerevisiae Atm1p is able to transport [Fe₂S₂](GS)₄ clusters [3]. Atm1p is a homolog of the human inner-mitochondrial membrane protein ABCB7. Recently the crystal structure of N. aromaticivorans Atm1p was solved with two oxidized glutathione molecules bound in the transport domain. The orientation of the oxidized glutathione molecules resembles the expected structure of the [Fe₂S₂](GS)₄ cluster. Mutations in the binding pocket, among other areas, in ABCB7 result in X-linked sideroblastic anemia [4]. This may be caused by the inability of the protein to transport [Fe₂S₂](GS)₄ clusters out of the mitochondria and into the cytosol.

References:
[1] Roualt, T.A. (2012) Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease, Dis Model Mech. 5(2), 155–164.
[2] Miao, R., Kim, H., Koppolu, U.M.K., Ellis, E.A., Scott, R.A., and Lindahl, P.A. (2009) Biophysical Characterization of the Iron in Mitochondria from Atm1p-depleted Saccharomyces cerevisiae, Biochemistry 48(40), 9556–9568.
[3] Qi, W., Li, J., and Cowan, J.A. (2014) A structural model for glutathione-complexed
iron–sulfur cluster as a substrate for ABCB7-type transporters, Chem. Commun. 50, 3795-3799.
[4] Lee, J.Y., Yang, J.G., Zhitnitsky, D., Lewinson, O., and Rees, D.C. (2014) Nature 343, 1133-1137

Keywords: Iron-Sulfur Cluster, Glutathione , ABC Transporter

21. Structural characterization of base excision repair processes catalyzed by polymerase β

Andrew J. Reed (OSBP), Rajan Vyas (Chem and Biochem), E John Tokarsky (Biophysics)

Abstract:
8-oxoG is the most common base modification and is generated via oxidation of the C8 position of guanine bases. While 8-oxoG can form a canonical Watson-Crick anti-8-oxoG:anti-dC base-pair, a new hydrogen bonding group on the Hoogsteen face allows for a syn-8-oxoG:anti-dA base-pair. Repair of 8-oxoG requires a minimum of four enzymes beginning with the actions of a glycosylase and endonuclease that generate a single-nucleotide gap flanked by a 3ʹ-OH and an unusual 5ʹ- dRP termini. This is followed by human DNA polymerase Beta (Pol Beta) catalyzed gap-filling DNA synthesis and 5ʹ-dRP removal. Finally, a human ligase seals the DNA backbone to terminate short patch BER. Alternatively, repair of an 8-oxoG:A mispair requires a glycosylase that excises the undamaged adenine. However, this requires Pol Beta to catalyze synthesis opposite 8-oxoG. In addition, if the dRP moiety is inefficiently removed in either instance, long patch BER is triggered wherein Pol Beta catalyzes strand displacement and DNA synthesis to incorporate one or more additional nucleotides followed by DNA processing by several other enzymes to complete DNA repair. Previous structural studies of Pol Beta and 8-oxoG have used substrate analogs that give only static images of pre-catalytic and post-catalytic by-pass of the lesion. We utilize time-resolved X-ray crystallography with natural substrates, to show lesion by-pass and extension in real time. In these structures, both base pairing modes are clearly visible. Surprisingly, a transient third metal is observed in the by-pass and extension reactions, and is hypothesized to aid in transition-state stabilization and product release. Furthermore, we utilize a unique Schiff-base reduction technique to capture Pol Beta in the process of removing a 5′-dRP. These structures provide an in depth examination of all the steps catalyzed by Pol Beta during short and long patch BER of 8-oxoG.

Keywords: DNA Polymerase, Oxidative Damage, X-ray Crystallography

22. Structural diversity at the interacting N-terminal tips of two non-classical cadherins.

Michelle E. Gray (Ohio State Biochemistry Program, The Ohio State University), Zachary R. Johnson (Department of Chemistry and Biochemisty, The Ohio State University), Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

23. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

Brandon L. Crowe (Ohio State Biochemistry Program), Ross C. Larue (College of Pharmacy, The Ohio State University), Chunhua Yuan (Campus Chemical Instrument Center, The Ohio State University), Sonja Hess (Proteome Exploration Laboratory, California Institute of Technology), Mamuka Kvaratskhelia (College of Pharmacy, The Ohio State University), Mark P. Foster (The Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Retroviruses must integrate their reverse-transcribed DNA into host chromatin in order to replicate. Sites of retroviral integration are not random and vary by retroviral genera. For example, γ-retroviruses such as murine leukemia virus (MLV) target transcription start sites. This preference is mediated through interactions between retroviral integrases (IN) and a host transcription factor protein family the bromodomain and extraterminal domain (BET) epigenetic reader proteins. The BET family contains two N-terminal bromodomains, which bind acetylated lysines that mark histones at transcription start sites, and an extraterminal (ET) domain which normally functions to recruit epigenetic modifying enzymes to chromatin. MLV IN binds the ET domain through a short conserved C-terminal motif we have named the ET binding motif (EBM). We used isothermal titration calorimetry (ITC) to show a tight interaction between the short MLV IN EBM and the Brd4 ET domain. We then used NMR to determine the solution structure of the Brd4 ET domain in complex with the MLV IN EBM. The structure reveals the formation of a three stranded antiparallel β-sheet, with two strands from the MLV IN EBM and one strand formed by structuring a loop in the ET domain. The structure reveals the formation of a hydrophobic core on one face of the β sheet and electrostatic interactions on the other face. Lastly, we have mutated the key residues on the Brd4 ET domain and studied the effect of these mutations on the ability of Brd4 ET to bind other transcription factors. Our data strongly suggests that the interaction characterized here mimics the cognate interactions relevant for the recruitment of other human transcription factors.

References:
Larue, R. C., Plumb, M. R., Crowe, B. L., Shkriabai, N., Sharma, A., Difiore, J., Malani, N., Aiyer, S. S., Roth, M. J., Bushman, F. D., Foster, M. P., and Kvaratskhelia, M. (2014) Bimodal high-affintiy association of Brd4 with murine leukemia virus integrase and mononucleosomes. Nucleic acids research. 42(8): 4868-81. doi. 10.1093/nar/gku135.

Crowe, B.L., Larue, R.C., Yuan, C., Hess, S., Kvaratskhelia, M., Foster, M.P. (2016) Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction. Proc. Natl. Acad. Sci. USA. 113(8): 2086-91. doi. 10.1073/pnas.1516813113.

Keywords: Retroviruses, BET transcription factor family, NMR solution structure

24. A novel mechanism of toxicity amplification: ACD toxin-produced actin oligomers poison formin controlled actin polymerization

David B Heisler (Ohio State Biochemistry Program and the Department of Chemistry and Biochemistry), Elena Kudryashova, Dmitry O. Grinevich (Department of Chemistry and Biochemistry, The Ohio State University), Cristian Suarez, Jonathan D. Winkelman (Department of Molecular Genetics and Cell Biology, The University of Chicago), Konstantin G. Birukov, Sainath R. Kotha (Section of Pulmonary and Critical Care and Lung Injury Center and Lipid Signaling and Lipidomics Laboratory, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine,), Dimitrios Vavylonis (Department of Physics, Lehigh University), David Kovar (Departments of Molecular Genetics and Cell Biology and of Biochemistry and Molecular Biology, The University of Chicago)

Abstract:
Bacterial toxins are the deadliest compounds on the planet; a single copy is capable of compromising a host cell. Amplification of toxicity is typically achieved by enzymatically targeting signaling cascades or inhibiting vital host proteins found in relatively few copies. Conversely, the major cytoskeletal protein actin is a common target for toxins, and it is not clear how actin-targeting toxins achieve high efficiency. The actin crosslinking domain (ACD) catalyzes the formation of an amide bond between actin monomers, forming actin oligomers. It was believed that ACD toxicity stems from the slow failure of the cytoskeleton due to the gradual accumulation of these non-functional actin oligomers. We found that ACD is not required to crosslink all actin in the cell; suggesting that low doese of ACD-crosslinked oligomers are toxic. Since actin-binding domains of actin-regulatory proteins are organized in tandems, these proteins can potentially bind actin oligomers with abnormally high affinities. Formins, a family of actin binding proteins, nucleate filaments and, through their tandem poly-proline stretches, bind profilin-actin complexes to accelerate filament elongation. We found that formins preferentially bind crosslinked oligomers and are inhibited by sub-nanomolar concentrations of oligomers as revealed on the single filament level by total internal fluorescence reflection microscopy. In the presence of profilin, the oligomers caused reversible blocks of elongation of formin-controlled filaments. Mathematical modeling revealed that the oligomers potently inhibit both nucleation and elongation of formin-controlled actin assembly. Our findings indicate that ACD employs a novel toxicity mechanism by converting actin into highly toxic oligomers to targeting key regulators of actin dynamics.

Keywords: Actin, Toxins, Cell biology

25. Using clathrin growth rates to demonstrate endocytic dynamics in vivo

Joshua Ferguson (Department of Physics, The Ohio State University), Nathan Willy, Scott Huber, Spencer Heidotting (Department of Physics, The Ohio State University), Matthew Webber (Biophysics Graduate Program, The Ohio State University), Comert Kural (Department of Physics, The Ohio State University)

Abstract not available online - please check the printed booklet.

26. Fhit loss - associated neoplasia initiation and progression in vitro

Jenna R. Karras (MCDB), Morgan S. Schrock (Biomedical Sciences), Bahadir Batar (MVIMG, The Ohio State University)

Abstract:
Genomic alterations are first observed at common fragile sites in preneoplastic cells, with FRA3B being the most frequently affected locus [1,2]. The FHIT gene encompasses FRA3B, resulting in the frequent loss or reduction of Fhit protein expression in the early stages of tumorigenesis. Loss of Fhit protein expression causes a nucleotide TTP pool imbalance via TK1 down-regulation, followed by replication stress – induced DNA damage. This damage accumulates with cell division in vitro, as Chk1 is not activated leading to global genome instability [3]. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. The goal of this study was to follow this process from loss of Fhit genome caretaker function, through development of genetic alterations to isolation of clones with tumorigenic potential. We established epithelial cell lines from kidney tissues of Fhit-/- and +/+ mice early after weaning of pups, and subjected some cell lines to nutritional and carcinogen stress; the Fhit +/+ cell lines did not survive either of these additional stresses. We confirmed that supplementation of the Fhit-/- cell lines with low levels of thymidine in culture medium prevented the accumulation of DNA damage. Through transcriptome profiling and protein expression analysis, we observed changes in the p53/p21 pathway as well as epithelial to mesenchymal transition states. Additionally, some of the Fhit-deficient cell lines demonstrated anchorage-independent growth and increased invasion capacity in vitro. When injected subcutaneously and intravenously into nude mice, cells of a stressed Fhit-/- cell line formed subcutaneous tumors and lung micro-metastases that expressed Vimentin, but not Cytokeratin or E-cadherin. Thus, Fhit loss-induced genome instability results in changes in expression patterns promoting cellular transformation in vitro and in vivo.

References:
1. Gourgoulis, V.G.; Vassiliou, L.V. Nature, 2005, 434, 907-913.
2. Bartkova, J.; Horejsi, Z. Nature, 2005, 434, 864-870.
3. Saldivar, J.C.; Miuma, S. PLoS Genet, 2012, 8, e1003077.

Keywords: common fragile site, genome instability, cell transformation

27. The uncharacterized fission yeast protein Rng15 aids efficient vesicle fusion at the division site required for late-stage cytokinesis

Kenneth S. Gerien (OSBP), Yihua Zhu (Department of Molecular Genetics, The Ohio State University), Jian-Qiu Wu (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

28. Ultrastructural Imaging of Collagen Fibrils in Mouse Model of Abdominal Aortic Aneurysm

Jeffrey R Tonniges (Biophysics, Ohio State University), Benjamin Albert (Biomedical Engineering, Ohio State University), Advitiya Mahajan (Center for Cardiovascular Research, Nationwide Childrens Hospital), Edward P Calomeni (Pathology, Ohio State University), Chetan P Hans (Center for Cardiovascular Research, Nationwide Childrens Hospital), Gunjan Agarwal (Biomedical Engineering, Ohio State University)

Abstract:
Abdominal aortic aneurysms (AAA) are a leading cause of death in the US. An aortic aneurysm is a persistent abnormal dilatation of the aorta that usually has no symptoms until it ruptures, which is a serious medical emergency. The specific cause of aortic aneurysms is still enigmatic, but it is believed to be initiated by degradation of elastin in the aortic wall. This degradation leads to weakening of the aorta, and in response the aorta is remodeled by degradation and deposition of collagen.

An open question in AAA pathology is: what is the quality of the collagen deposited in the AAA remodeling process and is this quality related to the risk of rupture. Previous studies of collagen in AAA have found defects in the organization and micro-architecture of collagen within AAA, and have correlated these defects with differences in aortic stiffness. However, only limited ultrastructural information has been gathered of collagen within AAA. This study aimed to determine if AAA contains ultrastructural defects in collagen.

To investigate the ultrastructural features of collagen in AAA, we imaged collagen in situ from a mouse model of AAA using transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Abnormal collagen fibrils with compromised banded structure were observed in AAA. A subset of abnormal collagen fibrils showed the normal ~67 nm D-banded structure, but contained a reduced depth of D-banded structure.

To explore a possible cause of the abnormal collagen fibrils, we examined the expression and phosphorylation levels of the collagen receptors, discoidin domain receptors (DDR1 and DDR2) using immunohistochemistry. DDR1 and DDR2 expression and phosphorylation staining was increased compared to control aortas. Most strikingly, there was little to no staining for DDR1 or DDR2 phosphorylation in control aortas, but strong staining in AAA, particularly in regions of collagen remodeling.

In conclusion, the abnormal collagen fibrils and correlation to increased DDR1 and DDR2 phosphorylation represent novel parameters to characterize pathological collagen remodeling in the arterial wall. Furthermore, the abnormal collagen fibrils could be a risk factor for aneurysm rupture, and DDR1 and DDR2 phosphorylation could be a therapeutic target for AAA.

Keywords: collagen, aneurysm, receptor tyrosine kinase

29. Identification of an inner nuclear membrane protein that impacts the spatio-temporal content of both the nuclear envelope and nuclear interior

Mahesh Chemudupati (Ohio State Biochemistry Program), Aysha Osmani (Department of Molecular Genetics, The Ohio State University), Stephen A. Osmani (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

30. Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

Yajun Liu (Department of Molecular Genetics), I-Ju Lee (Department of Pediatric Oncology, Dana-Farber Cancer Institute), Mingzhai Sun (Department of Surgery, Davis Heart and Lung Research Institute), Casey Lower (Department of Molecular Genetics), Jianjie Ma (Department of Surgery, Davis Heart and Lung Research Institute), Jian-Qiu Wu (Department of Molecular Genetics)

Abstract not available online - please check the printed booklet.

31. Bga1 is a novel regulator of β-glucan synthase in fission yeast

Reshma Davidson (MCDB), Josef Pontasch (Molecular Genetics), Jian-Qiu Wu (Molecular Genetics)

Abstract not available online - please check the printed booklet.

32. Title not available online - please see the printed booklet.

Qian Shi (MCDB), Paul K. Herman (Molecular Genetics)

Abstract not available online - please check the printed booklet.

33. Using Physarum polycephalum as a model organism for studying histone proteins inherent cell penetrating properties

Cecil J Howard II (Chemistry and Biochemistry), Angela Harrison (Chemistry and Biochemistry), Jennifer J Ottesen (Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

34. HAT1 orchestrates topological structure of chromatin during replication-coupled chromatin assembly.

Paula A Agudelo Garcia (MCDB), Michael Hoover (Department of Molecular Virology, Immunology and Medical Genetics), Michael Freitas (Department of Molecular Virology, Immunology and Medical Genetics), Mark Parthun (Biological Chemistry and Pharmacology)

Abstract not available online - please check the printed booklet.

35. Medicago truncatula LINC complexes and their potential role in root symbioses

Anna Hare Newman Griffis (Graduate Program in Molecular, Cellular and Developmental Biology, The Ohio State University), Katherine Beigel (Department of Molecular Genetics, The Ohio State University), Iris Meier (Department of Molecular Genetics, The Ohio State University)

Abstract:
Several vital crop plants’ productivity depends on root symbionts that provide the plants with nitrogen, phosphorus, and other nutrients. One of the most important events in the establishment of symbiosis is the initiation of interactions between plant roots and soil-dwelling microbes, including rhizobia and arbuscular mycorrhizal fungi. The role of nuclear migration in these processes has long been implied, but never definitively established. Recently, LINC complexes have been shown to be components of metazoan nuclear movement machinery. These complexes are made up of the inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins. While SUN proteins are widely conserved, no animal KASH protein homologs exist in plants. Working in the model plant Arabidopsis thaliana, our lab recently discovered the first plant KASH proteins, which are involved in processes ranging from nuclear movement in root hairs and pollen tubes to oomycete defense. However, Arabidopsis is a host to neither rhizobia nor arbuscular mycorrhizal fungi. Therefore, to study the role of LINC complexes in nuclear movement during the establishment of root-symbiont interactions, we have adopted a reverse genetics approach in the model legume Medicago truncatula. We have identified 12 genes that encode putative Medicago LINC complex components via bioinformatic tools. Here, we will show subcellular localization data for these LINC complex candidates from transient expression assays in Nicotiana benthamiana. Further candidate validation by co-immunoprecipitation with appropriate putative binding partners from Arabidopsis and Medicago (i.e. SUN-KASH interactions) will also be presented. Tnt1 retrotransposon insertion mutant lines have been obtained for 8 of the 12 putative LINC complex component genes. Results from phenotypic analyses performed on homozygous mutant lines, focusing on potential symbiosis and/or nuclear movement defects, will also be shown.

Keywords: plant-microbe interactions, cell biology, nuclear movement

36. Genetic Ablation of Smoothened in Pancreatic Fibroblasts Augments Acinar-Ductal Metaplasia Induced by Oncogenic Kras

Xin Liu (Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA), Jason R. Pitarresi (Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA), Jinghai Wu (Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA)

Abstract:
The contribution of the microenvironment to pancreatic acinar-to-ductal (ADM) is currently unclear. Here we show that disruption of paracrine hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a KrasG12D genetically engineered mouse model accelerated ADM formation. ADM was induced by activation of epithelial growth factor receptor (EGFR) through increased transforming growth factor alpha (TGF-) production in SMO-deleted fibroblasts. Increased expression and activity of the GLI family zinc finger protein 2 (GLI) transcription factor was the primary means of enhanced TGF- in fibroblasts. Upstream of TGF-, SMO-depleted fibroblasts exhibited activated p-AKT, which led to direct phosphorylation and stabilization of GLI2. These results define a non-cell autonomous mechanism that increased KrasG12D-driven ADM when hedgehog signaling is disrupted in stromal fibroblasts.

Keywords: pancreatic cancer , Hedgehog signaling

37. 3D inflation strains in porcine corneas

Keyton L. Clayson (Biophysics), Elias R. Pavlatos (Biomedical Engineering), Jun Liu (Biomedical Engineering, Ophthalmology, Biophysics)

Abstract not available online - please check the printed booklet.

38. Title not available online - please see the printed booklet.

Xiaoli Liu (WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States.), Jun Zhang (WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States.), Preethi Subramanian (WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States), Ramaswamy Bhuvaneswari (Dept. of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, United States), Knopp V. Michael (WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States)

Abstract not available online - please check the printed booklet.

39. Title not available online - please see the printed booklet.

Nathan Jones (OSU Biophysics), Miguel Lopez (Department of Molecular Virology, Immunology and Medical Genetics), Jeungphill Hanne (Department of Molecular Virology, Immunology and Medical Genetics), Jong-Bong Lee (Department of Physics, Pohang University of Science and Technology POSTECH), Richard Fishel (Department of Molecular Virology, Immunology and Medical Genetics), Kristine Yoder (Department of Molecular Virology, Immunology and Medical Genetics)

Abstract not available online - please check the printed booklet.

40. Title not available online - please see the printed booklet.

Pearson T. Maugeri (Biophysics Graduate Program), Effie K. Miller (Ohio State Biochemistry Program), Nicholas E. Trivelas (Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

41. Topological and statistical analyses of gene regulatory grids reveal unifying emergent properties

Wilberforce Zachary Ouma (Molecular Cellular and Developmental Biology Graduate Program), Mohammadmahdi R Yousefi (Department of Electrical and Computer Engineering, Ohio State University ), Erich Grotewold (Department of Molecular Genetics, Ohio State University )

Abstract:
Gene regulatory grids (GRGs) are static representations of gene regulatory networks (GRNs) encompassing all possible transcription factor (TF)-target gene interactions that provide a system-wide view of transcriptional gene regulation. To understand their architectural organization, transcriptional GRGs of Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae were constructed from experimental data and GRG emergent topological and statistical properties investigated. We examined the GRG degree connectivity by developing and implementing a formal statistical approach for fitting node degree to a power-law function. We observed that the out-degree, but neither the in- nor total-degree distribution, can be estimated by a power-law function. Unexpectedly, the exponent parameter of the power-law (alpha) was different for different organisms. In addition, the sampling of sub-grids of various sizes showed that exponents were generally invariant, thus providing us with a powerful tool to mathematically estimate the number of interactions that characterize the fully connected grids for these three organisms.
We hypothesized that a consequence of the scale-free property in cellular networks is faster signal propagation. To test this, we performed computational simulations to model the rate of signal propagation in GRGs and demonstrated that signals propagate faster in GRGs compared to their respective randomized grids. These observations support the hypothesis that network architecture and topology determines function, and that transcriptional GRGs have evolved to reduce the time taken for a signal to propagate throughout the grid.

Keywords: Gene regulatory grids , Network properties , Transcription

42. A multi-platform approach for the analysis of shotgun proteomics data

Owen E. Branson (The Ohio State Biochemistry Program), Michael A. Freitas (Department of Molecular Virology, Immunology and Medical Genetics)

Abstract:
The rapid development of mass spectrometry technologies has solidified shotgun proteomics as the most powerful analytical platform for large-scale proteome interrogation. The ability to map and determine differential expression profiles of the entire proteome is the ultimate goal of shotgun proteomics. Our novel approach to determine differential expression from spectral counts in shotgun proteomics leverages multiple statistical platforms that are routinely used to analyze RNA sequencing data. To remove bias associated with a single statistical approach, a single ranked list of differentially expressed proteins is assembled by means of comparing edgeR and DESeq q-values directly with the false discovery rate calculated by baySeq. Finally this statistical approach is then extended to spectral count data derived from multiple proteomic pipelines. The individual protein identifications and their respective statistics from multiple proteomic pipelines are integrated by collapsing protein groups across proteomic pipelines providing a single ranked list of differentially expressed proteins. Our approach of leveraging multiple search engines allows for in-silico cross-validation of proteomic results and increases the depth of the experiment with the ultimate goal of understanding the biological system of interest.

Keywords: Proteomics, Mass Spectrometry

43. Gene expression changes in late-onset Alzheimer's Disease

Carol J. Huseby (Interdisciplinary Biophysics Progrm), Lauren Lin (Undergraduate Neuroscience Program), Connor S. Wagner (Undergraduate Neuroscience Program), Jeff Kuret (Biological Chemistry and Pharmacology, Wexner Medical Center)

Abstract not available online - please check the printed booklet.

44. Modeling the Response of Cardiac Troponin C to Calcium on the Thin Filament: Effects of Disease-Related and Post-Translational Modifications

Jalal Siddiqui (Biophysics), Bin Liu (Physiology and Cell Biology), Shane Walton (Biophysics), Vikram K. Shettigar (MCDB), Andrew J. ONeil, Peeyush Shrivastava, Nathan Neilson, Grace Davis (Physiology and Cell Biology), Svetlana Tikunova and Brandon Biesiadecki (Physiology and Cell Biology)

Abstract:
Calcium binding to and dissociation from cardiac troponin C (TnC) are essential steps leading to cardiac muscle contraction/relaxation. It is well documented that the calcium binding properties of TnC are not constant, but are sensitive to complex interactions between the additional thin and even thick filament proteins. There is a growing body of evidence that protein modifications/mutations within different subunits of the troponin complex (troponin T, troponin I) exert their effects by altering the apparent TnC calcium sensitivity/exchange kinetics. There are a number of potential mechanisms that could alter the calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. We have developed a rather simple mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We propose that roughly half of the studied modifications do not alter any of the intrinsic TnC calcium binding constants but rather alter the ability of TnC to “find” TnI in the presence of calcium. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium and that TnI also binds to actin, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC.

Keywords: Cardiac Muscle, Troponin , Mathematical Model

45. RNMT catalyzes cytoplasmic mRNA cap methylation and is a component of the cytoplasmic capping complex

Jackson B. Trotman (Ohio State Biochemistry Program, The Ohio State University), Chandrama Mukherjee (Department of Biological Chemistry and Pharmacology, The Ohio State University), Daniel R. Schoenberg (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract not available online - please check the printed booklet.

46. Newly Introduced Transposable Elements are Silenced by Expression-Dependent RNA directed DNA Methylation

Dalen R. Fultz (MCDB), R. Keith Slotkin (Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

47. Title not available online - please see the printed booklet.

Katherine M. McKenney (Ohio State Biochemistry Program, Department of Microbiology and OSU Center for RNA Biology), Ian M.C. Fleming (Department of Microbiology and OSU Center for RNA Biology), Kirk W. Gaston (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Pat A. Limbach (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology), Juan D. Alfonzo (Department of Microbiology and OSU Center for RNA Biology)

Abstract not available online - please check the printed booklet.

48. Title not available online - please see the printed booklet.

Randal J. Soukup (MCDB), Richard A. Fishel (Molecular Virology, Immunology and Medical Genetics)

Abstract not available online - please check the printed booklet.

49. Title not available online - please see the printed booklet.

Kiel D. Kreuzer (MCDB), Nicholas J. Green (MCDB), Frank J. Grundy (Department of Microbiology), Tina M. Henkin (Department of Microbiology)

Abstract not available online - please check the printed booklet.

50. Investigating the molecular basis for substrate recognition by Thg1

Ashanti Matlock (OSBP), Jane Jackman (Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

51. Understanding EGF pathway interaction differences in nematodes using SUR-2/MED23

Karley K. Mahalak (Molecular, Cell, and Developmental Biology), Abdulrahman M. Jama (Molecular Genetics, OSU), Steven Billups (Molecular Genetics, OSU), Edward Zitnik (Molecular Genetics, OSU), Helen M. Chamberlin (Molecular Genetics, OSU)

Abstract not available online - please check the printed booklet.

52. PAX protein coordination of cell fate and organogenesis in Caenorhabditis elegans

Allison Webb (OSBP), Ryan Johnson, Helen Chamberlin (OSU Molecular Genetics)

Abstract not available online - please check the printed booklet.

53. Investigating the function of the muscle-specific fusion regulator Myomaker in skeletal muscle fusion and homeostasis

Kimberly J. Hromowyk (MCDB ), Jared C. Talbot (Postdoc, Molecular Genetics, The Ohio State University), Sharon L. Amacher (P.I.)

Abstract not available online - please check the printed booklet.

54. Genomic profiling reveals novel transcriptomes and temporal dynamics of murine heart valve endothelial cells

Lindsey J Anstine (1Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA,2Center for Cardiovascular Research, The Research Institute at Nationwide C), Blair F Austin (2Center for Cardiovascular Research, The Research Institute at Nationwide Childrens Hospital, Columbus, OH, USA ), Tori Horne (The Ohio State University, Columbus, OH, USA.), Joy Lincoln (2Center for Cardiovascular Research, The Research Institute at Nationwide Childrens Hospital, Columbus, OH,3Department of Pediatrics, The Ohio State University, Columbus, OH, USA)

Abstract:
Heart valve insufficiency affects ~2.5% of the population and aging is a significant risk factor, however its not clear why. In the elderly, composition of the valve extracellular matrix (ECM) is altered, and studies have shown that valve interstitial cells (VICs) mediate ECM homeostasis, while overlying valve endothelial cells (VECs) regulate VIC function. Based on this, we hypothesize that age-dependent changes in endothelial cell dynamics underlie valve disease in the aging population. Using electron microscopy, we observe changes in VEC morphology and distribution in aging valves. In addition using RNA-seq, we have generated age-dependent molecular profiles of VECs isolated from mice at embryonic (E14.5), post-natal (PN), adult (4 months) and aged (12-15 months) stages. Additional analysis identified Focal-Adhesions as a significantly affected pathway between embryonic, post-natal and adult time points. We validated this finding by testing VEC permeability in vivo using Evans Blue and found VECs in aged mice are more permeable than those of weanlings, suggesting a potential mechanism by which the aging valve is more susceptible to systemic age related changes. As cell cycle genes were markedly reduced in VECs following post-natal growth and endothelial stem cell markers were detected, we examined if circulating cells play a role in maintaining the adult valve endothelium. To do this, we performed bone marrow transplants and observe donor cells co-expressing endothelial markers on the surface of the valve cusps. Current studies are investigating the functional differences between young and aging VECs as well as the contribution and molecular plasticity of bone marrow-derived cells engrafted within young and aging valves.

Keywords: heart valve, aging

55. Title not available online - please see the printed booklet.

James Boslett (MCDB)

Abstract not available online - please check the printed booklet.

56. Single-molecule investigation of response to oxidative DNA damage by a Y-family DNA polymerase

Austin Raper (Ohio State Biochemistry Program), Varun Gadkari (Ohio State Biochemistry Program), Brian Maxwell (Ohio State Biophysics Graduate Program)

Abstract:
Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium toward a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG.

Keywords: polymerase, single-molecule, fluorescence

57. Characterization of Function and Mechanism of Recognition of an Orphan 3'-5' RNA Polymerase.

Samantha Dodbele (Ohio State Biochemistry Program), Yicheng Long (Ohio State Biochemistry Program), Jane Jackman (Ohio State Biochemistry Program)

Abstract not available online - please check the printed booklet.

58. Kinetic characterization of the human DNA polymerase ε holoenzyme

Walter J. Zahurancik (OSBP)

Abstract:
Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'→5' exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits with no known catalytic activity that associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of Polɛ from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of human Polɛ (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not significantly affect the nucleotide incorporation rate constant or nucleotide binding affinity. Furthermore, the small subunits do not appear to affect the rate-limiting step of correct nucleotide incorporation. Together, these data suggest that the role of the small subunits in vivo is primarily limited to mediating protein-DNA and protein-protein interactions. Importantly, this study provides the framework for future kinetic investigation of the impact of the other proteins known to interact with Polε at the replication fork.

Keywords: DNA polymerase, pre-steady-state kinetics, DNA replication

59. Title not available online - please see the printed booklet.

Jonathan W. Picking (The Ohio State Biochemistry Program, The Ohio State University), Duncan J. Kountz (Department of Microbiology, The Ohio State University), Edward J. Behrman (Department of Chemistry & Biochemistry, The Ohio State University), Joseph A. Krzycki (Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

60. Title not available online - please see the printed booklet.

Adil Moghal (The Ohio State Biochemistry Program and the Department of Microbiology, The Ohio State University), Lin Hwang (Pasarow Mass Spectrometry Laboratory and the Semel Institute of Neuroscience and Human Behavior, University of California at Los Angeles), Kym Faull (Pasarow Mass Spectrometry Laboratory and the Semel Institute of Neuroscience and Human Behavior, University of California at Los Angeles), Michael Ibba (The Ohio State Biochemistry Program and the Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

61. Function of the m1G9 tRNA modification and its catalyst, Trm10

Nathan Howell (OSBP), Abigail Hubacher (Chemistry and Biochemistry), Aiswarya Krishnamohan (OSBP)

Abstract not available online - please check the printed booklet.

62. Title not available online - please see the printed booklet.

Jack Tokarsky (Ohio State Biophysics Program), Petra Wallenmeyer (Ohio State Chemistry Program), Kenny Phi (Department of Chemsitry and Biochemistry)

Abstract not available online - please check the printed booklet.

63. Dynamics and mechanism of cyclobutane pyrimidine dimer repair by Class II DNA photolyase and the role of Adenine in repair

Meng Zhang (Biophysics Graduate Program, The Ohio State University), Zheyun Liu (Department of Chemistry and Biochemistry, The Ohio State University), Shi Shu (Department of Chemistry and Biochemistry, The Ohio State University), Lijuan Wang (Department of Physics, The Ohio State University), Aziz Sancar (Department of Biochemistry and Biophysics, University of North Carolina School of Medicine), Dongping Zhong (Department of Physics, Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Photolyase utilizes blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD) by splitting the cyclobutane ring. In our previous studies we resolved the complete repair photocycle by Class I photolyase with E. coli photolyase as a model system and concluded that the adenine moiety of the unusual bent flavin cofactor is essential to mediating the electron transfer between lumiflavin and CPD through a superexchange mechanism. Here we further studied CPD repair by Class II photolyase with femtosecond resolution and strikingly, we found that in Class II photolyase from Arabidopsis thaliana (AtCPD), the electron transfer adopts a direct two-step hopping process between lumiflavin and CPD bridged by adenine. The first-step hopping from the lumiflavin to adenine occurs in 560 ps, and the second-step hopping from the adenine moiety to substrates takes tens of picoseconds with a short distance of 3.1 Å. In photolyases from Drosophila, Methanosarcina mazei, and in the single-strand DNA photolyase from Arabidopsis thaliana, electron transfer from lumiflavin to adenine and to CPD have comparative probabilities and a mixed mechanism of electron tunneling and hopping was proposed. The cyclic intramolecular electron transfer between lumiflavin and adenine was observed in all photolyase, with a relatively fast backward ET ranging from 10 ps to 400 ps. The intrinsic cyclic ET in photolyases plays a critical role in mediating electron transfer in repair of damaged DNA through a superexchange or a two-step hopping mechanism.

Keywords: DNA repair, electron transfer, ultrafast spectroscopy

64. Polarity and Charge as determinants for translocase requirement for Membrane protein insertion.

Balasubramani Hariharan (Biophysics Graduate Program), Raunak Soman (OSBP)

Abstract:
Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. It constitutes for one third of the cellular proteome. The two key components discovered so far in this cascade of events from protein translation to being inserted into the inner membrane are the Sec translocon complex (SecYEG) and Membrane protein insertase YidC. Both the proteins are universally conserved and translocate about 95% of the membrane proteins. YidC functions both independently and in concert with the Sec translocon.(1) The crystal structure revealed the presence of a hydrophilic groove open to both the cytoplasm and the lipid bilayer. Structure guided biochemical assays unveiled a mechanism for single transmembrane protein translocation which involves interaction of charged residue in residue in the hydrophilic groove of YidC.(2) The mechanism for larger substrates is not yet understood completely. We hypothesize that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we have tested this polarity/charge hypothesis by showing that Major coat protein of bacteriophage M13 (Gene VIII) can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent substrates. Additionally we test whether the length of procoat loop and the positive charge are determinants for Sec dependence and find that as we increase the length of the loop it becomes more YidC/Sec independent. We picture the insertion of substrates as a phenomenon happening at the interface of both YidC and Sec translocon. These properties can be greatly utilized when designing the epitope for efficient Phage display that can be used as a therapeutic agent.(3) This study also throws light on the requirements of both the insertase to coexist in all walks of life.

Keywords: Membrane Protein Insertion, Phage display, Sec Translocon

65. Defining the role of PAK7 variants in melanoma

Kyle M LaPak (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Michael. A. Gross (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Denny C. Vroom (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Greg B. Lesinski (Department of Medical Oncology and Microbial Infection & Immunology, The Ohio State University, Columbus, OH 43210), William E. Carson (Department of Molecular, Virology, Immunology, & Medical Genetics and Surgical Oncology, The Ohio State University, Columbus, OH 43210), Christin E. Burd (Department of Molecular Genetics and Molecular, Virology, Immunology, & Medical Genetics, The Ohio State University, Columbus, OH 43210)

Abstract not available online - please check the printed booklet.

66. RCAN1-4 Regulates Thyroid Cancer Growth and Metastasis

Chaojie Wang (Ohio State Biochemistry Program), Saji Moto (Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine), Steven E Justiniano (Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine), Adlina Mohd Yusof (Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine), Matthew D Ringel (Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine)

Abstract:
Metastatic progression is the main cause of thyroid cancer deaths. Metastasis suppressors are key regulators of tumor invasion and metastases. Loss of metastasis suppressors has been associated with aggressive tumor behaviors and metastatic spread. We previously showed that Regulator of Calcineurin 1, isoform 4 (RCAN1-4) was upregulated by a metastatic suppression pathway and could inhibit cell motility when overexpressed in thyroid cancer cells. Here, we hypothesized RCAN1-4 was a metastasis suppressor in vivo. Using novel RCAN1-4 knockdown stable cells, we showed that knocking down RCAN1-4 in cancer cells promoted cell invasion without affecting cell proliferation. Subcutaneous tumor models demonstrated that RCAN1-4 knockdown in tumor cells increased tumor growth rate and tumor volumes. Metastasis models showed that RCAN1-4 knockdown promoted tumor metastases to the lungs. Microarray analysis discovered Nuclear Factor, Erythroid 2-Like 3 (NFE2L3) was a pivotal downstream effector of RCAN1-4 in regulating tumor metastasis. TCGA thyroid cohort data also demonstrated that NFE2L3 expression was significantly higher in the tumor samples compared with normal tissues. In conclusion, we provided the first evidence that RCAN1-4 was a tumor metastasis suppressor in thyroid cancer cells in vivo.

Keywords: RCAN1-4, Thyroid Cancer, Metastasis

67. Title not available online - please see the printed booklet.

Jason R. Pitarresi (OSBP), Xin Liu (OXBP), Jinghai Wu (OSU Comprehensive Cancer Center)

Abstract not available online - please check the printed booklet.

68. Investigations of fibrotic pathway activation downstream from the mineralocorticoid receptor in a Duchenne muscular dystrophy model.

J. Spencer Hauck (Department of Physiology and Cell Biology, OSU Wexner Medical Center, Ohio State University, Columbus OH), Callie Brown, Jessica Chadwick, Jill A. Rafael-Fortney

Abstract not available online - please check the printed booklet.

69. Proof-of-Principle Study Shows Efficient Skipping of Exon 2 Using Antisense Morpholino Oligomers

Tabatha R. Simmons (Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH), Nicolas Wein (Center for Gene Therapy, The Research Institute at Nationwide Childrens, Columbus, OH), Felecia Gumienny (Center for Gene Therapy, The Research Institute at Nationwide Childrens, Columbus, OH), Hannah Huang (Center for Gene Therapy, The Research Institute at Nationwide Childrens, Columbus, OH), Kevin M. Flanigan (Center for Gene Therapy, The Research Institute at Nationwide Childrens, Columbus, OH; Departments of Pediatrics and Neurology, The Ohio State University, Columbus, OH)

Abstract:
Currently, exon skipping therapies for Duchenne muscular dystrophy (DMD) have been developed for patients with out-of-frame deletions where treatment will lead translation of an internally truncated but partially functional dystrophin protein. In contrast, we are focusing on treating duplications mutations, accounting for around 6% of all mutations, resulting in wild-type transcript and a full-length protein. Modeling the most common single exon duplication we have developed the first duplication mouse containing a duplicated exon 2. We have performed a proof-of-principle study using antisense oligomer (AO)-induced exon 2 skipping using this Dup2 mouse. Intramuscular (TA) injections of exon 2-directed different antisense peptide-morpholino conjugates (PPMO) were performed at either 10 or 20 ug total PPMO (N=6 muscles each) doses. Mice were injected at 8 weeks and sacrificed 1 month later for muscle analysis of DMD mRNA and dystrophin protein expression. Additionally, systemic (tail vein) injections are being conducted of at a dose of 30 ug/kg, at 1 week, 2 week and 1 month timepoints. For the IM studies, a gradient of exon 2 exclusion was seen by RT-PCR at both doses with corresponding high levels of properly localized dystrophin protein by IF and western blot.
Analyses of RT-PCR and protein expression are underway for the systemic delivery PPMOs. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping - to the exclusion of exon 2 entirely - results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

Keywords: DMD, Antisense, Skipping

70. Inhibition of liver inflammation and tumorigenesis by blocking the Ccl2-Ccr2 axis in a mouse model

Kun-Yu Teng (Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH), Jianfeng Han (Comprehensive Cancer Center, The Ohio State University, Columbus, OH), Xiaoli Zhang (Center of Biostatistics, The Ohio State University, Columbus, OH), Linda A. Snyder (Janssen Research and Development, LLC, Spring House, PA), Jianhua Yu (Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH), Shu-hao Hsu, Kalpana Ghoshal (Pathology, The Ohio State University, Columbus, OH)

Abstract not available online - please check the printed booklet.

71. Stromal PDGFR-alpha activation stalls mammary ductal development and renders the epithelium more tumorigenic

Anisha M. Hammer (MVIMG), Gina M. Sizemore (MVIMG), Vasudha Shukla (Biomedical Engineering ), Cecilia Cuitino (MVIMG), Samir Ghadiali (Biomedical Engineering), Michael Ostrowski (MVIMG)

Abstract not available online - please check the printed booklet.

72. Title not available online - please see the printed booklet.

Tchekneva, E.E., Akhter, A. (Department of Internal Medicine), Antonucci, A.E. (Biophysics Program and Department of Internal Medicine), Evans, J.V. (Department of Internal Medicine), Long, N.E., Campbell, C.R., Callahan, N.W., Magliery, T. (Department of Chemistry and Biochemistry), Carbone, D.P., and Dikov, M.M. (Department of Internal Medicine)

Abstract not available online - please check the printed booklet.

73. The CSF1-PU.1 Pathway in Tumor Associated Macrophages Promotes Breast Cancer Growth and Progression

Katie Thies (MCDB), Haritha Mathsyaraja, David A. Taffany, Sudarshana Sharma (Molecular Virology, Immunology & Medical Genetics, The Ohio State University), W. Hans Meisen, Balveen Kaur (Department of Neurological Surgery, The Ohio State University), Tom Liu, Cynthia Timmers (The Comprehensive Cancer Center, The Ohio State Unviersity), Jose Otero (Department of Pathology, The Ohio State University), Michael C. Ostrowski (Molecular Virology, Immunology & Medical Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

74. Title not available online - please see the printed booklet.

Pooja Joshi (MCDB), Young-Jun Jeon (Stanford University)

Abstract not available online - please check the printed booklet.

75. The role of microRNA in chemoresistance in glioblastoma

Theresa Relation (Neuroscience Graduate Program), Ji Young Yoo (Department of Neurological Surgery, The Ohio State University), Chelsea Bolyard-Blessing (Department of Neurological Surgery, The Ohio State University), Cristina Jaime-Ramirez (Department of Neurological Surgery, The Ohio State University), Selene Virk (Department of Epidemiology and Biostatistics, Case Western Reserve University), Jill Barnholtz-Sloan (Department of Epidemiology and Biostatistics, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

76. The Role of Chronic Inflammatory Environment in promoting Breast Cancer Progression

Tasha Wilkie (MCDB), Mohd Nasser (University of Nebraska Medical Center), Helong Zhao (University of Utah)

Abstract not available online - please check the printed booklet.

77. Epigenetic classification of CLL using the novel Bisulfite-iPLEX technique

Brian J. Giacopelli (Molecular, Cellular, and Developmental Biology), Yue-Zhong Wu (Comprehensive Cancer Center, The Ohio State University), John C. Byrd (Department of Internal Medicine, Division of Hematology, The Ohio State University), Christopher C. Oakes (Department of Internal Medicine, Division of Hematology, The Ohio State University)

Abstract:
Subclassification of tumor samples using epigenetic patterns is an emerging and powerful approach to predict clinical outcomes and improve therapy selection. Current technology for analyzing DNA methylation ranges from global assays, which provide a large amount of data but at a high cost per sample, to targeted assays, which cost less per sample but focus on a single small region of DNA. To simultaneously address numerous genomic regions in a high-throughput and cost efficient manner, a new approach was needed. We developed a novel method for analyzing the methylation status of multiple CpGs named Bisulfite-iPLEX (Bs-iPLEX) using the MassARRAY system (Agena Biosciences) which utilizes a MALDI-TOF mass spectrometer to analyze oligonucleotides. This method is capable of screening up to 30 individual CpGs multiplexed in a single well in 384 samples simultaneously. Here we demonstrate that the Bs-iPLEX is a highly accurate, reproducible, and efficient tool for measuring DNA methylation.
As an application of the Bs-iPLEX, we designed a panel of targeted CpGs to profile the epigenetic pattern of chronic lymphocytic leukemia (CLL) patients. CLL is a clinically heterogeneous disease which can be classified into three subgroups based on their epigenetic profile defined by genome-wide DNA methylation patterns using 450K arrays (Illumina). Our multiplexed panel accurately classifies CLL patient subgroups using a greatly reduced number of CpGs (20 CpGs) defferentially methylated among CLL patients. We initially evaluated the accuracy of the panel using a set of 96 patient samples of known subgrouping. We further validated with a set of an additional 96 patient samples. We conclude that the Bs-iPLEX is an efficient and accurate technique for assaying the DNA methylation profile of patient samples, and can effectively be used to classify CLL patients with potential clinical utility.

Keywords: Epigenetics, Cancer

78. The microbiome in lung and breast cancer: An RNA-seq analysis

Carmine M. Sonzone (OSU, MCDB Program), Xiaodan Mai (Univserity at Buffalo, Epidemiology and Environmental Health), Daniel Weng, Catalin Marian (OSUCCC), Max Westphal, Alex Pelletier, Angela Urdaneta, Paige Stump, Cameron Stump (OSUCCC Genomics Shared Resource), Pearlly Yan, Ralf Bundschuh (OSUCCC Genomics Shared Resource, OSU Division of Hematology), Jean Wactawski-Wende, Jo Freudenheim (Univserity at Buffalo, Epidemiology and Environmental Health)

Abstract:
There is increasing evidence that the microbiome profoundly affects human health and disease, including cancer. There is separately substantial data for the role of inflammation in carcinogenesis, which we postulated is related to alterations in the microbiome of normal tissues and tumors. The presence of bacteria in breast and lung cancers has been documented, including by us, but a comprehensive analysis of the microbiome has not been done, nor a linkage to chronic inflammation. It is our hypothesis that specific bacteria or a change in the pattern of bacteria (dysbiosis) induce chronic inflammation that leads to breast and lung carcinogenesis. For the lung, bacteria enter the lung through direct inhalation while smoking. For the breast, there are multiple ways that bacteria will reach the breast, including blood dissemination from the oral cavity and gastrointestinal tract, or retrograde entry while breast feeding. The goal of this study is to confirm the presence of bacteria in lung (n=30) and breast cancer (n=30) as well as adjacent normal tissue, demonstrate that bacteria can be found in lung (n=10) and breast tissue (n=10) of healthy individuals without cancer, and relate the bacterial presence to inflammation. We will identify which bacteria are also present in the oral cavity, GI tract or tobacco. Our data demonstrate that there are numerous live bacteria present, and a prioritization scheme has identified at least 16 potential pathogens in breast cancers and 27 potential pathogens in lung cancers. Justification for the oral cavity as a potential sight of origin for these tumor bacteria is the observation that a majority of the bacteria identified by our prioritization scheme have been identified by others as normal flora of the oral cavity. This study is innovative in both testing a novel hypothesis for breast and lung cancer etiology and use of state-of-the-art next generation RNA-sequencing. The significance of this study is the potential to identify an entirely new avenue of investigation for breast and lung cancer risk. The presence of bacteria, as well as dysbiosis, and an understanding of the associations with inflammation could provide new methods and markers for prevention, prognosis and treatment (e.g., a differential effect by new immunotherapies and pathways stimulated by bacterial infections).

Keywords: RNA-seq, Cancer, Microbiome

79. Targeting cyclin-dependent kinases in triple negative breast cancer

Douglas G. Cheung (Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University), Gianpiero di Leva, Matteo Fassan, Claudia Piovan (Department of Molecular Immunology, Virology and Medical Genetics, The Ohio State University), Krishna Patel, Arpan Kumar, Dorothee Wernicke, Stefano Volinia (Department of Molecular Immunology, Virology and Medical Genetics, The Ohio State University), Marina Ciomei (Business Unit Oncology, Nerviano Medical Sciences), Michela Garofalo (Transcriptional Networks in Lung Cancer, CRUK Manchester Institute), Carlo M. Croce (Department of Molecular Immunology, Virology and Medical Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

80. Identification of Biomarkers Associated with MEK Inhibitor GSK1120212 Mediated Increase in Thyroidal Radioactive Iodine Uptake in a Mouse Model of Papillary Thyroid Cancer.

Daniel Scarberry (Molecular, Cellular, and Developmental Biology), Sissy Jhiang (Physiology and Cell Biology, College of Medicine)

Abstract not available online - please check the printed booklet.

81. Parental exposure to dim light at night prior to mating alters offspring adaptive immunity

Yasmine M. Cisse (Department of Neuroscience, Behavioral Neuroendocrinology Group, The Neuroscience Research Institute, The Ohio State University Wexner Medical Center, Columbus OH 43210), Randy J. Nelson (Department of Neuroscience, Behavioral Neuroendocrinology Group, The Neuroscience Research Institute, The Ohio State University Wexner Medical Center, Columbus OH 43210)

Abstract:
Environmental light is the most potent signal for synchronizing the circadian system. Adoption of electrical lighting has occurred without understanding the physiological effects of light at night (LAN). Disruption of natural light/dark cycles by LAN dampens endogenous biological rhythms that maintain optimal function of various systems, including the immune system. Dim LAN (dLAN) exposure impairs innate and cell mediated immune responses in Siberian hamsters (Phodopus sungorus). Studies on the effects of circadian disruption have thus far focused on adults, but impaired maternal immune and endocrine function has downstream effects on offspring immune phenotype. The circadian system exerts an organizational role in these systems and dLAN exposure impairs their function. Thus, we hypothesized that parental exposure to dLAN prior to mating impairs offspring immune function. Adult male and female Siberian hamsters were exposed to either dark nights (DA) or dLAN (DI) for 9 weeks, at which point they were paired, mated, and thereafter housed in dark nights. Pairings resulted in four groups: DA/DA (Male/Female), DA/DI, DI/DA, and DI/DI. Adult offspring were tested for cell-mediated, humoral and innate immunity. Male offspring of dams exposed to dLAN suppressed cell-mediated swelling reactions. Male offspring of DA/DI and DI/DA parents produced more antibodies in response to a novel antigen. In female offspring, maternal and paternal exposure to dLAN decreased swelling response. Female offspring of dams exposed to dLAN produced more antibodies in response to challenge. No deficits in plasma bactericidal capacity were observed. Overall, parental exposure to dLAN decreased offspring cell-mediated immunity and enhanced humoral immunity in a parent and offspring sex-specific manner. Altered immune responsiveness in offspring that have experienced dLAN in the germline indicates that seemingly innocuous nighttime lighting may have transgenerational effects on immune function.

Keywords: circadian disruption, immunity, transgenerational

82. Restraint stress induces plasticity and regeneration in peripheral neurons

Kathryn M Madalena (Neuroscience), Akhil Dhamija, Ash Zawerton, Harrison Taylor, Jessica K Lerch (Neuroscience)

Abstract:
Restraint stress induces plasticity in the central nervous system (Vyas et al., 2002) and increases mechanical allodynia after spared nerve injury (Alexander et al., 2009). We have replicated the findings that acute stress (one hour, one time) increases mechanical allodynia. We expanded this investigation using chronic stress (two hours per day for two weeks) and found the exact opposite effect. Chronic stress ameliorated the increased allodynia. The evidence for changes in the nervous system led us to study the effects of restraint stress on growth in dorsal root ganglia (DRG) sensory neurons and regeneration in the sciatic nerve. We find that restraint stress, both acute and chronic, significantly increased neurite outgrowth in DRG neurons measured after 24 hours of growth in vitro. Regeneration in the sciatic nerve can be observed at 1-3 days post crush injury using the marker for regenerating axons SCG10 (Shin et al., 2014). One hour of restraint stress given immediately prior to crush injury increased regeneration in the sciatic nerve three days post injury compared to non-stressed injured mice. We conclude from these findings that restraint stress can induce growth in DRGs and regeneration in the sciatic nerve. We expect that the changes observed are mediated by a glucocorticoid/glucocorticoid receptor mechanism and are working to develop mice with a conditional deletion of the glucocorticoid receptor in sensory neurons.

References:
Alexander JK, DeVries AC, Kigerl KA, Dahlman JM, Popovich PG (2009) Stress exacerbates neuropathic pain via glucocorticoid and NMDA receptor activation. Brain Behav Immun 23:851–860

Shin JE, Geisler S, DiAntonio A (2014) Dynamic regulation of SCG10 in regenerating axons after injury. Exp Neurol 252:1–11

Vyas A, Mitra R, Shankaranarayana Rao BS, Chattarji S (2002) Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J Neurosci 22:6810–6818.

Keywords: Glucocorticoids, Stress, Plasticity

83. Enhanced synaptogenesis in autonomic circuitry following spinal cord injury

Benjamin Noble (Neuroscience Graduate Program), Masaki Ueno (Cincinnati Childrens Hospital), Cagla Eroglu (Duke University), Yutaka Yoshida (Cincinnati Childrens Hospital), Phillip G. Popovich (Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University. )

Abstract:
Autonomic dysreflexia (AD) is a potentially fatal complication of spinal cord injuries (SCI) occurring at or above the fifth thoracic vertebral level. The hallmark symptom of AD is unchecked activation of the spinal autonomic (sympathetic) reflexes. This can lead to various complications such as sweating, immune suppression, and cerebral hemorrhage. The mechanisms responsible for the onset and progression of AD are not well defined, but since supraspinal brain connections are mostly lost after high-level SCI, maladaptive plasticity within “presympathetic” neurons (neurons synapsing on the sympathetic preganglionic neurons of the autonomic nervous system) is expected. We hypothesize that (1) the increased frequency of spontaneous AD is caused by enhanced excitatory synaptogenesis on sympathetic preganglionic neurons (SPNs) within the intermediolateral nucleus (IML), (2) synapse number will increase as a function of time post-injury correlating with an increase in AD frequency, and (3) silencing the excitatory sympathetic circuitry will prevent the development of AD and the resulting complications. The overall number of synapses was quantified using confocal microscopy and automated quantification programs. Our data reveal an increase in synapses over time, reaching maximal numbers at 28 days post injury, a time coinciding with the peak number of daily dysreflexic episodes, suggesting a direct link between post-SCI synaptogenesis and AD. To silence this newly developed circuitry, we are utilizing Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). DREADDs are specially designed receptors that can be targeted to a specific subtype of cells. Though preliminary, pilot studies have shown that the silencing of this circuitry can ameliorate certain sequelae of AD, such as immune suppression, measured by spleen size and immune cell quantification. These findings may identify a potential method of therapy for post-SCI patients suffering from AD.

References:
Zhang, Y., et al. Autonomic dysreflexia causes chronic immune suppression after spinal cord injury. J Neurosci 33, 12970-12981 (2013).

Lucin, K.M., Sanders, V.M., Jones, T.B., Malarkey, W.B. & Popovich, P.G. Impaired antibody synthesis after spinal cord injury is level dependent and is due to sympathetic nervous system dysregulation. Exp Neurol 207, 75-84 (2007).

Meisel, C., Schwab, J.M., Prass, K., Meisel, A. & Dirnagl, U. Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci 6, 775-786 (2005).

Brommer B, Engel O, Kopp MA, Watzlawick R, Müller S, Prüss H, Chen Y, DeVivo MJ, Finkenstaedt FW, Dirnagl U, Liebscher T, Meisel A, Schwab JM. Spinal cord injury-induced immune deficiency syndrome enhances infection susceptibility dependent on lesion level. Brain. 2016 Mar;139(Pt 3):692-707.

Keywords: Spinal Cord Injury, Autonomic Dysreflexia, Neuroplasticity

84. High-thoracic spinal cord injury alters the activity and function of hematopoietic stem & progenitor cells

Randall S. Carpenter (Neuroscience Graduate Program, The Ohio State University, Columbus, OH), Phillip G. Popovich (Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University, Columbus, OH)

Abstract not available online - please check the printed booklet.

85. Gene therapy for Rett syndrome

Samantha Powers (Department of Neuroscience, Ohio State University), Carlos Miranda (Center for Gene Therapy, Nationwide Childrens Hospital), Lyndsey Braun (Center for Gene Therapy, Nationwide Childrens Hospital), Amy Huffenberger (Center for Gene Therapy, Nationwide Childrens Hospital), Kathrin Meyer (Center for Gene Therapy, Nationwide Childrens Hospital), Brian Kaspar (Center for Gene Therapy, Nationwide Childrens Hospital; Department of Neuroscience, Ohio State University)

Abstract:
Rett syndrome is a devastating and progressive neurodevelopmental autism spectrum disorder (ASD) affecting approximately 1 in 10,000 girls. Rett patients experience loss of achieved developmental milestones including speech and motor function beginning at around 12-18 months of age. Patients typically live to middle age; however, they require significant supportive measures including 24/7 care. This produces a significant emotional and financial burden on patients and families. Rett syndrome is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2), a ubiquitous transcription factor with activation and repression functions for thousands of genes in the brain. Recent studies using rodent genetic models of MeCP2 demonstrated that Rett syndrome-like phenotypes, including autism-like behavior and shortened lifespan, could be ameliorated by the re-expression of MeCP2. This lead the field of Rett syndrome research to pursue research avenues to augment MeCP2 expression in Rett patients and potentially other ASD cases. Our laboratory has pioneered the field of gene therapy using adeno-associated virus (AAV) to treat neurodegenerative disorders, with current clinical trials ongoing in spinal muscular atrophy (SMA) type 1 and Batten’s disease. We are currently evaluating a gene therapy approach for Rett syndrome. We have generated an AAV serotype 9 (AAV9) vector expressing the human MeCP2 gene under the control of its endogenous promoter. When administered systemically, AAV9 has the ability to cross the blood brain barrier and transduce neurons and astrocytes. We are currently evaluating our vector to identify the proper route of delivery (systemic versus direct brain injection) and identifying the minimal effective dose. Preliminary results show good levels of expression of our vector upon a single injection. More importantly, they show a significant positive impact in Rett syndrome-like behavioral phenotypes and survival on MeCP2 knockout mice. Completion these studies will pave the way for further studies with non-human primates in order to lead our work from the bench to the bed side.

Keywords: Rett syndrome, Gene Therapy, AAV9

86. HSPB1-Mediated Non-Cell Autonomous Neuroprotection of Motor Neurons is Disrupted by Mutations that Result in Hereditary Neuropathy

Patrick L. Heilman (The Ohio State Biochemistry Program), Dr. SungWon Song, Dr. Carlos Miranda (The Institute for Gene Therapy), Dr. Kathrin Meyer (The Institute for Gene Therapy), Dr. Brian K. Kaspar (The Institute for Gene Therapy), Dr. Stephen J. Kolb (Department of Biological Chemistry and Pharmacology, Department of Neurology)

Abstract:
Objective: To study the neuroprotective effects of wild type and disease associated mutant HSPB1 in an established in vitro ALS co-culture model system.

Methods: HSPB1 lenti-viral constructs were transduced into wild type and ALS murine NPC-derived astrocytes. Astrocytes were then co-cultured with murine motor neurons in vitro, with motor neuron survival being measured daily.

Results: Our results show that HSPB1 overexpression in SOD1-G93A astrocytes promotes motor neuron survival in vitro. By day 6, HSPB1 expression increased motor neuron survival by 10-fold compared to astrocytes expressing SOD1-G93A alone. Overexpression of phosphomimetic HSPB1 (HSPB1-TriD) resulted in a 40% increase in motor neuron survival. Overexpression of mutant HSPB1 resulted in a reduction of neuroprotection (HSPB1-R136W) or in a complete loss of neuroprotection (HSPB1-G84R). siRNA-mediated knockdown of endogenous HSPB1 had had no effect on motor neuron survival.

Conclusion: We conclude that non-cell autonomous overexpression of HSPB1 protects motor neurons from SOD1-mediated toxicity in vitro and this protection may be mediated by HSPB1 phosphorylation. Further, mutations in HSPB1 associated with late on-set distal hereditary motor neuropathy fail to protect motor neurons in this model system. Finally, we show that astrocyte-specific expression of these HSPB1 mutants are themselves toxic to motor neurons.

Keywords: HSPB1, Motor Neuron Disease, Molecular Chaperone

87. Discrepancy Between Behavioral and Electrophysiological Measurements in a Longitudinal Study of Motor Unit Recovery

Christopher G. Wier (Neuroscience Graduate Program), Kajri Sheth (Department of Biological Chemistry and Pharmacology), Patrick L. Heilman (The Ohio State Biochemistry Program), W. David Arnold M.D. (Department of Neurology, Department of Neuroscience, Department of Physical Medicine and Rehabilitation), Stephen J. Kolb M.D.,Ph.D. (Department of Neurology, Department of Biological Chemistry and Pharmacology)

Abstract:
Approximately 20 million Americans suffer from a peripheral nerve injury, with a majority occurring from traumatic causes. Following injury, peripheral nerves will eventually regenerate and reinnervate their target muscle fibers unlike injuries to the central nervous system. Pre-clinical functional behavior assays—toe spread measurements and toe spread reflexes—are used to record regeneration of the motor unit (MU) in animal models. Electrophysiological markers—compound muscle action potential (CMAP), motor unit number estimate (MUNE) and fibrillations with electromyography (EMG)—have been used by clinicians to diagnose patients with MU diseases in addition to tracking peripheral nerve recovery in patients. Pre-clinical longitudinal studies comparing the recovery rates of behavioral and electrophysiological measures are needed for understanding the underlying mechanisms of MU recovery. For this study, a complete sciatic nerve injury in adult mice was utilized to longitudinally compare the rates of MU recovery in behavioral functional assays and a previously described protocol for electrophysiological markers. Behavioral measures returned to baseline by 28 days post injury (28dpi) in all mice. EMG post-crush showed prominent fibrillation potentials (gastrocnemius), but by 28dpi minimal fibrillations were noted in only 23%. In contrast, at 28 dpi, CMAP and MUNE were 48% (18.1 ±7mV) and 29% (103 ±40 motor units) recovered. CMAP recovered to 74% (28.3 ±9mV) of baseline, within the standard deviation of the baseline measurement at 42 dpi, but MUNE was only 43% recovered (155 ±45 motor units). By 60 dpi, MUNE was still only 63% recovered (226 ±59 motor units). This suggests that mechanisms involved in motor unit recovery are still ongoing regardless of apparent behavioral recovery. Motor neuron quantification was investigated and axon pathology is on-going to provide an underlying mechanism. To better determine the effectiveness of pre-clinical therapeutics for motor unit recovery, electrophysiological markers should be used alongside functional behavioral assays during the time course of study.

Keywords: Motor Unit, Peripheral Nerve Injury, Electrophysiology

88. Central and Peripheral Immune Signaling at the Brain Endothelium is Crucial in Stress-induced Anxiety-like Behavior

Anzela Niraula (Neuroscience Graduate Program, Institute for Behavioral Medicine Research), Daniel McKim (Neuroscience Graduate Program, Institute for Behavioral Medicine Research), Caroline Sawicki (College of Dentistry, Institute for Behavioral Medicine Research), Brant Jarrett (Institute for Behavioral Medicine Research, College of Dentistry), Dr. Jonathan Godbout (Neuroscience Graduate Program, Institute for Behavioral Medicine Research), Dr. John Sheridan (College of Dentistry, Institute for Behavioral Medicine Research)

Abstract not available online - please check the printed booklet.

89. Region specific inflammatory responses remote to thoracic spinal cord injury

Timothy D. Faw, PT, DPT, NCS (Neuroscience Graduate Program, The Ohio State University), Diana M. Norden, PhD (Center for Brain and Spinal Cord Repair, The Ohio State University), Rochelle J. Deibert (Center for Brain and Spinal Cord Repair, The Ohio State University), Lesley C. Fisher (School of Health and Rehabilitation Sciences, The Ohio State University), D. Michele Basso, EdD, PT (School of Health and Rehabilitation Sciences, The Ohio State University)

Abstract:
Spinal cord injury (SCI) produces a toxic inflammatory microenvironment that negatively affects plasticity and recovery. Recently, we showed microglial activation, infiltration of bone marrow-derived myeloid cells, and increased cytokine production in the remote lumbar cord within the first 24hrs after thoracic SCI (T9). At the same time, cervical regions were protected from this robust central and peripheral inflammatory response. Importantly, inflammation and myeloid cell infiltration may impede exercise-based recovery. The purpose of this study was to characterize the regional specificity of inflammatory responses in remote spinal cord regions after thoracic SCI. Mice received a 75kdyn contusion at T9 using the Infinite Horizons device with inflammation measured at cervical and lumbar sites 1-14 days later. Within 24hrs, CD11b+/CD45high myeloid cells infiltrated the lumbar but not cervical cord. Lumbar but not cervical localization of GFP+ bone marrow derived myeloid cells using chimeric mice confirmed regional differences. Further, quantitative PCR identified a pro-inflammatory profile of lumbar infiltrating macrophages at 24hrs with increased expression of inflammatory cytokines (TNFα), chemokines (CCL2), and adhesion molecules (ICAM). The number of peripheral monocytes in the lumbar cord was attenuated by 14 days. Conversely, central signs of inflammation and gliosis (Iba-1, GFAP) remained increased at 14 days only in the lumbar cord. These data suggest that a toxic microenvironment driven by central and peripheral immune responses develops in the remote lumbar cord after SCI. Interestingly, the cervical cord is protected from inflammation which may render it uniquely capable of adaptive plasticity early after SCI when the lumbar microenvironment renders training ineffective. Future studies will determine the cervical response to neurorehabilitation after SCI.

References:
Hansen, C. N., Fisher, L. C., Deibert, R. J., Jakeman, L. B., Zhang, H., Noble-Haeusslein, L., et al. (2013). Elevated MMP-9 in the Lumbar Cord Early after Thoracic Spinal Cord Injury Impedes Motor Relearning in Mice. Journal of Neuroscience, 33(32), 13101–13111. http://doi.org/10.1523/JNEUROSCI.1576-13.2013

Detloff, M. R., Fisher, L. C., McGaughy, V., Longbrake, E. E., Popovich, P. G., & Basso, D. M. (2008). Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Experimental Neurology, 212(2), 337–347. http://doi.org/10.1016/j.expneurol.2008.04.009

Keywords: Spinal Cord Injury, Inflammation, Remote

90. Sex differences in the effects of microglia on neonatal neurogenesis and behavioral development

Lars H nelson (Neuroscience Graduate Program), Spencer Warden (Department of Psychology), Kathryn M. Lenz (Department of Psychology and Neuroscience)

Abstract not available online - please check the printed booklet.

91. Identification of Modifiers of Spinal Muscular Atrophy

Corey Ruhno (OSBP), Vicki McGovern (Biological Chemistry and Pharmacology), Jesse Hunter (TGen), Lisa Baumbach-Reardon (TGen), Matthew Hudson (University of Illinois), John McPherson (Ontario Institute of Cancer Research)

Abstract not available online - please check the printed booklet.

92. Identification and Characterization of Novel Tax-1 Interacting Protein, SNX27, and its Role in HTLV-1 Pathobiology

Jacob Al-Saleem (Center for Retrovirus Research, Department of Veterinary Biosciences, College of Veterinary Medicine,The Ohio State University, Columbus, OH, USA), Nikoloz Shkriabai (Center for Retrovirus Research, Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH, USA), Mamuka Kvaratskhelia (Center for Retrovirus Research, Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH, USA), Lee Ratner (Division of Oncology, Washington University, St Louis, MO, USA), Patrick L. Green (Center for Retrovirus Research, Department of Veterinary Biosciences, College of Veterinary Medicine, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Col)

Abstract not available online - please check the printed booklet.

93. High-throughput RNA 3D motif structure prediction and validation

Jian Wu, Cuiji Zhou, Ying Wang, James Li, Dana Driver (Dept. of Molecular Genetics, Center for Applied Plant Sciences, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Neocles Leontis (Dept. of Chemistry, Bowling Green State University, Bowling Green, OH 43403), Craig Zirbel (Dept. of Mathematics and Statistics, Bowling Green State University, Bowling Green, OH 43403), David M. Bisaro (Dept. of Molecular Genetics, Center for Applied Plant Sciences, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Biao Ding (Dept. of Molecular Genetics, Center for Applied Plant Sciences, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
The genomic RNA and its derivatives of an RNA virus or subviral agent (a viroid, satellite RNA or satellite RNA virus) specifies all biological functions required to establish infection. These functions are accomplished through the encoded protein products or through the regulatory structural elements of the RNA itself. Because RNA-RNA or RNA-protein interactions critical for function are often mediated by local 3D motifs, a key to understand viral evolution and function is genome-wide knowledge of these motifs. X-ray crystallography and NMR have played a major role in deciphering the 3D structures of some viral RNA motifs, but the technical challenges render genome-wide determination of viral RNA local 3D motifs a daunting task. We use Potato spindle tuber viroid (PSTVd) as a model to develop a high-throughput platform for local 3D motif structure analysis, by combining homology-based structural prediction using existing structural information from the database, deep-sequenced viroid population information, and mutagenesis-based functional determination. With this approach we have predicted and validated the 3D structures of many PSTVd loops. We will present our data and discuss the broad implications of our approach.

Keywords: RNA, 3D sturucture, PSTVd

94. Mechanism of human lysyl-tRNA synthetase/tRNALys preimer recruitment and packaging into HIV-1

Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un), Nathan Titkemeier (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un), Corine St. Gelais (Department of Veterinary Biosciences, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Li Wu (Department of Veterinary Biosciences, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un)

Abstract:
The primer for reverse transcription in HIV-1, human tRNA^Lys3, is selectively packaged into virions along with tRNA^Lys1,2. We have previously reported that human lysyl-tRNA synthetase (hLysRS), the only cellular factor known to interact specifically with all three tRNA^Lys isoacceptors, is also packaged into HIV-1 and the presence of both host cell factors is required for optimal viral infectivity. How the hLysRS/tRNA^Lys complex is diverted from its normal cellular location into HIV-1 particles is unknown. hLysRS is normally part of a dynamic mammalian multisynthetase complex (MSC). In recent years, hLysRS has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways that involve nuclear localization, membrane binding, and secretion. Here, we show that the expression of hLysRS is unaltered upon HIV-1 infection of HEK293T and CD4+ HuT/CCR5+ T cells, suggesting that the hLysRS species packaged is recruited from an existing pool of LysRS. Immunofluorescence and confocal microscopy imaging reveal that hLysRS is released from the MSC and relocalized to the nucleus upon HIV-1 infection. Our studies also indicate that hLysRS is phosphorylated after HIV-1 infection. This post-translational modification results in release from the MSC and nuclear entry, as pre-treatment of cells with a MEK inhibitor known to block specific hLysRS Ser phosphorylation results in reduced nuclear localization and importantly, reduced HIV-1 infectivity. Taken together, these studies shed light on the mechanism of tRNA^Lys,3 primer recruitment into HIV-1 and suggest a new avenue for targeting a host cell factor that is essential for HIV-1 infection.

Keywords: aminoacyl-tRNA synthetase, HIV, LysRS

95. Title not available online - please see the printed booklet.

Tami Coursey (Molecular, Cellular, and Developmental Biology), Milica Milutinovic (Institute for Biological Research Sinia Stankovi, University of Belgrade), Jelena Brkljacic (Arabidopsis Biological Resource Center, Ohio State University), David Bisaro (Molecular Genetics, Ohio State University)

Abstract not available online - please check the printed booklet.

96. The Roles of RNA Polymerases II, IV and V in Defense Against Geminiviruses

Jessica Storer (Molecular Genetics and Molecular, Cellular and Developmental Biology Program, The Ohio State University), Jamie N. Jackel (Molecular Genetics, The Ohio State University), Tami Coursey (Molecular Genetics and Molecular, Cellular and Developmental Biology Program, The Ohio State University)

Abstract:
Epigenetic modifications are influential in organizing and controlling gene expression. One such example of epigenetic control is histone and cytosine methylation that leads to transcriptional gene silencing of potentially harmful DNA, such as transposable elements and viruses. Geminiviruses are a family of plant viruses that package small, circular, and single-stranded DNA genomes. These form double-stranded DNA intermediates that associate with histones to form minichromosomes. As such, these viruses are subject to, and repressed by, epigenetic modifications leading to transcriptional gene silencing. Arabidopsis encodes plant-specific RNA polymerases IV (Pol IV) and V (Pol V), currently thought to establish repressive cytosine methylation leading to transcriptional gene silencing. Using geminiviruses as a de novo model to more precisely define the role of Pol IV and Pol V in initiating chromatin methylation, we determined that these polymerases are required for the establishment of repressive dimethylated histone 3 lysine 9 (H3K9me2) marks. However, they are not required to initiate cytosine methylation, implicating another polymerase in this process. Pol IV and V are multi-subunit enzymes that are related to, and share subunits with, RNA polymerase II (Pol II), an enzyme that orchestrates the formation of heterochromatin and transcriptional gene silencing in S. pombe. We found that plants deficient for Pol II are hypersusceptible to geminivirus infection, and that Pol II is also required for establishment of H3K9me2 on the viral chromatin. In addition, Pol II is also required to recruit Pol IV to the viral genome. Thus Pol II is essential for establishment of a repressive histone mark, likely via recruitment of Pol IV. Its involvement in cytosine methylation is under investigation.

Keywords: RNA-directed DNA methylation, RNA polymerase II , geminivirus

97. The Apical Loop of the F2 Peptide in the Respiratory Syncytial Virus Fusion Protein Plays an Essential Role in Membrane Fusion

Stephanie N. Hicks (OSBP), Supranee Chaiwatpongsakorn (Vaccines and Immunity, NCH), Heather M. Costello (Vaccines and Immunity, NCH)

Abstract:


The RSV fusion (F) protein is produced in a metastable, prefusion conformation that refolds to the stable, postfusion conformation when triggered by an unidentified stimulus. This conformational change serves to mediate fusion between an F protein expressing membrane and an adjacent membrane. The prefusion F protein structure has been solved in complex with monoclonal antibody (mAb) D25 that specifically recognizes the prefusion form. The ability of D25 to stabilize the prefusion form and its high neutralizing activity suggests its apical epitope is important for protein function. We hypothesize that residues in this binding site, particularly those in the apical F2 loop, are required for F protein triggering and, therefore, its fusion function. Twelve residues in the apical loop of the F2 domain were mutated to assess their importance in fusion. The ability of these mutants to cause fusion was determined with a cell-cell fusion assay. Six of the alanine-scanning mutants were deficient in their ability to cause fusion, five of which were loss of charge mutants. Mutations conserving or altering each residues charge demonstrated the importance of the natural charge of each residue, and of the F2 apical loop, in general, for F protein function. Each mutant’s cell surface conformation was determined by their reactivity with mAbs specific to the prefusion or postfusion form. All six mutants remained in the pretriggered form but were capable of causing fusion under low molarity conditions. This result suggests that these mutants are not trapped in the prefusion conformation and may, instead, comprise the natural attachment or triggering site on the RSV F protein.

Keywords: RSV, Fusion Protein

98. Title not available online - please see the printed booklet.

Christine Wachnowsky (OSBP, Ohio State), Insiya Fidai (Biophysics, Ohio State), Nathaniel Wesley (Department of Chemistry and Biochemistry, Ohio State)

Abstract not available online - please check the printed booklet.

99. Aldosterone Synthase is Increased in Dystrophic Muscle

Jessica Chadwick (OSBP, The Ohio State University ), Sarah Swagger (Physiology and Cell Biology, The Ohio State University ), Jeovanna Lowe (Physiology and Cell Biology, The Ohio State University )

Abstract not available online - please check the printed booklet.

100. sHBEGF stimulates endogenous muscle expression of Galgt2 (B4Galnt2) and Galgt2-dependent genes known to inhibit muscular dystrophy

Megan L. Cramer (Molecular, Cellular, and Developmental Biology Graduate Program), Rui Xu, Guohong Shao, and Paul T. Martin (Center for Gene Therapy at The Research Institute at Nationwide Childrens Hospital)

Abstract not available online - please check the printed booklet.

101. Probing Nucleosome Stability with DNA Origami Calipers

Jenny V. Le (Interdisciplinary Biophysics Graduate Program), Yi Luo (Interdisciplinary Biophysics Graduate Program), Michael A. Darcy (Department of Physics, OSU), Christopher R. Lucas (Department of Mechanical and Aerospace Engineering, OSU), Michael G. Poirier (Department of Physics, OSU), Carlos E. Castro (Department of Mechanical and Aerospace Engineering, OSU)

Abstract:
While biophysical tools such as single molecule fluorescence and force spectroscopy have been used to study the structural dynamics of individual nucleosomes or large chromatin assemblies, it is challenging to probe conformational dynamics of gene regulation in the 10-100nm range. This is the length scale of genomic critical features, including promoter regions that span several nucleosomes. Our work aims to develop DNA origami tools to probe these mesoscale structure and dynamics of nucleosome arrays and chromatin. DNA origami itself is an emerging nanotechnology that enables the self-assembly of precisely designed molecular-scale structures. Here, we implement a DNA origami hinge device with the length scale of ~100nm as a nanocaliper to study the structure and dynamics of a single nucleosome as proof-of-concept.

The measurement capabilities of the device were calibrated by integrating DNA duplexes of varying lengths between the hinge arms to demonstrate the use the hinge angular distribution as a readout for the sample size attached between the arms. We further integrated single nucleosomes with varying but symmetric lengths of DNA linkers (i.e. DNA extending out after wrapping around the histone core) to verify our ability to detect structural changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosome-caliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements.

These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm.

Keywords: nucleosomes, structural dynamics, DNA origami

102. Modifying the stability of tumor suppressor p53 through S7-S8 loop mutagenesis and NMR studies of the pheromone Er-23

David Bowles (OSBP), David Rabinovich (Undergraduate), Sidharth Mohan (Biophysics), Calvin Rhoads (Undergraduate)

Abstract:
The tumor suppressor p53 is a critical 54 kDa protein responsible for the regulation of cellular differentiation during times of cellular stress. This protein is so critical is to be found to be mutated in approximately 50% of all human cancer cases. The majority of these mutations are found in the central, ordered DNA-binding domain of the protein. One way in which these mutations can disrupt DNA binding is to lower the overall stability of the protein, making it unable to properly interact with DNA. Consequently, any knowledge that can be gained in stabilizing the p53 DNA-binding domain could potentially be very valuable. It has been observed that the p53 homolog in C. elegans is considerably more stable than its human counterpart. By comparing the sequences of human p53 and worm p53, it was proposed that shortening the S7S8 turn is an important factor in this stability. This initial work was done in silico, and the Magliery lab is constructing mutants based on this hypothesis to be expressed and screened in vitro. By expressing and screening various four- and five- residue deletion mutants, insight was gained into which residues are important in the loop. These results will be used to design a loop mutant library that will probe sequence space for mutants that are more stable than the control construct. In addition to the worm p53 comparison strategy, algorithmic improvement of p53 has been attempted using methodology developing in the Magliery lab. The resulting construct is algorithm derived p53, or Algo p53, has been expressed and characterized. As a prelude to p53/CEP-1 NMR work, NMR work on a smaller protein has been started. Our lab has produced the pheromone ER-23 from E. coli cells, previously thought not viable. This allowed us to produce an N-15/C-13 labeled version from which to determine an NMR solution structure with more constraints than the natural abundance only structure in the literature.

Keywords: protein engineering, NMR, protein loops

103. Modeling dynamics of DNA origami structure

Abstract not available online - please check the printed booklet.

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