OSU Life Sciences Interdisciplinary Graduate Programs Symposium 2015

Keywords: R2lox, metal incorporation, resonance Raman spectroscopy

35. Dynamic determination of the functional state in photolyase

Meng Zhang (Biophysics Graduate Program, The Ohio State University), Zheyun Liu (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 is a photoenzyme which utilizes blue light to repair UV-damaged DNA. The catalytic cofactor of photolyase, flavin adenine dinucleotide, has an unusual bent configuration in photolyase, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.

Keywords: DNA repair, electron transfer, ultrafast spectroscopy

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

Xiaoli Liu (1WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States), Jun Zhang (1WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States), Bhuvaneswari Ramaswany (2Internal Medicine, Ohio State University Medical Center, Columbus, OH, United States), Michael V. Knopp (1WCIBMI, Dept. of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, United States)

Abstract not available online - please check the printed booklet.

37. Tau aggregation mechanism includes end-to-end annealing

Carol J. Huseby (Biophysics Graduate Program), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Center for RNA Biology), Jeff Kuret (Department of Molecular & Cellular Biochemistry, College of Medicine)

Abstract not available online - please check the printed booklet.

38. Shotgun proteomics reveal pathways pivotal in pre-neoplastic prostate development

Owen E. Branson (Ohio State Biochemistry Graduate Program, The Ohio State University), Michael E. Hoover (Biomedical Sciences Graduate Program, The Ohio State University), Jennifer M. Thomas-Ahner (Division of Medical Oncology, Department of Internal Medicine, The Ohio State University), Steven K. Clinton (Division of Medical Oncology, Department of Internal Medicine, The Ohio State University), Michael A. Freitas (Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

39. Electrokinetic effects of biomolecules: Methods and Application to DNA oligomer

Mithila V. Agnihotri (Biophysics Program, The Ohio State University), Si-Han Chen (Chemistry Department, The Ohio State University), Corey Beck (Chemistry Department, The Ohio State University), Sherwin J. Singer (Chemistry Department and Biophysics Program, The Ohio State University)

Abstract:
The goal of our work is to theoretically examine the dynamics of a DNA oligomer and its counter-ion atmosphere in response to a time varying electric field. These effects, termed electrokinetic effects, are of interest for technological reasons since electric fields can be harnessed to manipulate and transport biomolecules in a controlled fashion in micro- and nano-scale biomedical devices¹. Another motivation for this work stems from the need to interpret the results of dielectric spectroscopy, which is a commonly used probe of the properties of biomolecules and their surrounding electrical double layer. Our study of a DNA oligomer is motivated by recent experiments on short oligomers, the first time that data is available for oligomers small enough to be accessible to molecular dynamics simulations. One of the difficulties faced in these calculations is due to the large time scale associated with biomolecule motions. Hence one needs to accumulate very long trajectories, which requires large storage capabilities. We have re-formulated the theory in a way that enables calculation of transport properties from both equilibrium and non-equilibrium simulations using sparse sampling of data without compromising accuracy². We have shown proof-of-principle by calculating the flow velocity profile for electro-osmotic flow (a type of electrokinetic phenomenon) for a simple electrolyte system using both equilibrium and non-equilibrium simulations. We have also calculated the ionic contribution to frequency dependent electric susceptibility for the same system. Calculation of the electrokinetic properties of a double stranded DNA dodecamer are ongoing. Preliminary data are presented.

References:
[1] Yaling Liu, Qingjiang Guo, Shunqiang Wang, and Walter Hu. Electrokinetic effects on detection time of nanowire biosensor. Applied Physics Letters, 100(15):153502, April 2012.
[2] Mithila V. Agnihotri, Si-Han Chen, Corey Beck, and Sherwin J. Singer. Displacements, Mean-Squared Displacements, and Codisplacements for the Calculation of Nonequilibrium Properties. The Journal of Physical Chemistry B, 118(28):8170–8178, July 2014.

Keywords: DNA dynamics, Dielectric spectroscopy, molecular dynamics

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

Tang-Yu Liu (Biophysics program)

Abstract not available online - please check the printed booklet.

41. Alternative selection of TSSs between tissues and focused transcriptional initiation are a key features of maize core promoters

Maria Katherine Meja-Guerra (Center for Applied Plant Sciences CAPS, Molecular Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH 43210), Wei Li (Department of Physiology and Cell Biology, Heart and Lung Research Institute, Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Narmer Fernando Galeano, Mabel Vidal (Center for Applied Plant Sciences CAPS, The Ohio State University, Columbus, OH 43210), John Gray (6Department of Biological Sciences, University of Toledo, Toledo, OH), Andrea I. Doseff (Department of Physiology and Cell Biology, Heart and Lung Research Institute, Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Erich Grotewold (Center for Applied Plant Sciences CAPS, Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

43. Mass spectrometry-aided identification of substrate contact sites in a proteinaceous RNase P

Tien-Hao Chen (Chemistry and Biochemistry), Akiko Tanimoto (Chemistry and Biochemistry), Vicki Wysocki (Chemistry and Biochemistry), Venkat Gopalan (Chemistry and Biochemistry)

Abstract:
RNase P, which catalyzes tRNA 5’-maturation, is an essential enzyme that displays diversity in its make-up: a catalytic RNA-based ribonucleoprotein (RNP) form in all domains of life, and an RNA-free, protein-based version present only in certain eukaryotes. Because RNase P is unique in its ability to use either an RNA- or a protein-based active site for catalyzing the same reaction, understanding substrate recognition and catalysis by these variants might provide insights into the evolution of biocatalysts. Unlike the well-studied RNP form, the proteinaceous RNase P (PRORP) has drawn attention only recently. The crystal structure of PRORP reveals two motifs implicated in RNA processing: pentatricopeptide repeat (PPR) and metallonuclease (MN) domains. The goal of this study is to understand how PRORP uses these two domains to recognize and cleave a precursor tRNA (pre-tRNA) substrate. To identify the substrate-contacting residues, we incubated either the PRORP─pre-tRNA complex or PRORP alone with N-hydroxysuccinimide-biotin to modify solvent-exposed lysines, with the expectation that lysines contacting the pre-tRNA would be protected from modification. The differential accessibility of lysines was determined using proteolysis and mass spectrometry (MS). Our results revealed 86% of the lysines in PRORP are modified. Of these, we identified four pre-tRNA-contacting lysines in the PPR domain, and three in the MN domain. Mutating these lysine residues in either the PPR or MN domains resulted in binding or catalytic defects, respectively, and validated the MS-aided footprinting data. These results lead to two inferences about RNase P diversity and evolution. First, unlike the RNP, PRORP uses a smaller surface area to recognize pre-tRNAs, partly accounting for differences in catalytic potential between these variants. Second, both forms of RNase P appear to have evolutionarily converged on the theme of using distinct domains for substrate recognition and cleavage.

Keywords: RNA-Protein interaction, Mass Spectrometry, PRORP

44. Investigating the molecular basis for substrate recognition by Thg1

Ashanti Matlock (Ohio State Biochemistry Program), Jane E. Jackman (Ohio State Biochemistry Program)

Abstract not available online - please check the printed booklet.

45. Heat shock response alters SMN alternative splicing

Catey Dominguez (MCDB), Dawn Chandler (MCDB, The Research Institute at Nationwide Childrens)

Abstract not available online - please check the printed booklet.

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

Daniel F. Comiskey Jr. (MCDB), Aishwarya G. Jacob (MCDB), Ravi K. Singh (MCDB), Dawn S. Chandler (MCDB)

Abstract not available online - please check the printed booklet.

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

Kiel D. Kreuzer (MCDB, Department of Microbiology, Center for RNA Biology, The Ohio State University), Nicholas J. Green (MCDB, Department of Microbiology, The Ohio State University), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

48. Exploring endothelial cell dynamics in the development, maintenance and regeneration of heart valves

Lindsey J. Miller (Department of Pediatrics, The Ohio State University, Columbus, OH, USA;The Research Institute at Nationwide Childrens Hospital Department of Pediatrics, The Ohio State University, Columbus, OH, USA), Blair F. Austin (Center for Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Childrens Hospital), Joy Lincoln (Department of Pediatrics, The Ohio State University, Columbus, OH, USA;The Research Institute at Nationwide Childrens Hospital Department of Pediatrics, The Ohio State University, Columbus, OH, USA)

Abstract:
Heart valve insufficiency affects ~2.5% of the population with increasing incidence in the elderly. Studies have shown that valve dysfunction is caused by changes in the contribution and organization of extracellular matrix (ECM) that lead to biomechanical failure. Deposition and degradation of the ECM is mediated by valve interstitial cells (VICs) and data suggest that overlying valve endothelial cells (VECs) establish and maintain function of the VIC population. Therefore, we hypothesize that changes in endothelial cell dynamics underlie valve dysfunction. To address this, we developed a method to isolate VECs from Tie2-GFP mice using FACS analysis. With this approach we performed RNA-seq on VECs isolated from embryonic (E14.5), post-natal (PN), adult (4 months) and aged (15 months) mice and generated unique molecular profiles at each time point. Pathway analyses revealed VECs during embryonic and post-natal growth are highly proliferative and express pluripotency markers including telomerase (Tert) compared to adult VECs. However, although mRNA expression decreases, Tert activity remains high in adult stages. As it is unclear whether this pluripotent subpopulation is resident to the valve or arises from a satellite source, we are examining the contribution of bone marrow-derived (BM) progenitor cells to the valve and have data to show that BM cells incorporate into the VEC layer of adult mice. Ongoing studies will examine BM contribution to VECs in mice exposed to valve injury or disease. Together these results highlight the pluripotent potential of VECs after birth and will provide insight into the therapeutic potential of VECs for heart valve regeneration and repair.

Keywords: Heart valve, endothelial cell, regeneration

49. EGL-38/PAX functions in communicating cells of the Caenorhabditis elegans egg-laying system to coordinate signaling and differentiation

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

Abstract:
Paired-box (Pax) transcription factors are essential regulators of coordinated development responsible for controlling the growth and differentiation of diverse cells into organ systems. Pax transcription factors rely on cellular context to initiate their cell-specific activity through poorly understood mechanisms. To investigate PAX function, we are utilizing the PAX2/5/8 Caenorhabditis elegans ortholog EGL-38 as a simplified model system. EGL-38 functions in the hermaphrodite egg-laying system to coordinate anchoring of the vulva to the uterus. To initiate creation of this vulval-uterine connection, a LIN-3/Epidermal Growth Factor (EGF) signal is sent from the vulval vulF cells to the LET-23/EGFR receptor on a subset of neighboring uterine cells. These cells are specified as uv1 and are characterized by non-migration and by expression of the neuropeptide nlp-2. EGL-38 is required for lin-3/egf expression in the vulF cells ^(1,2), thereby initiating this signaling pathway. EGL-38 may function in response to the LIN-3/EGF pathway in the uv1 cells, as we have confirmed that EGL-38 can bind the nlp-2 promoter in vitro, and that both uv1 non-migration and nlp-2 expression are significantly decreased in egl-38 mutants as well as in lin-1 mutants. LIN-1 is an ETS-family transcription factor; ETS proteins often serve as PAX co-factors to confer DNA binding specificity ⁽³⁾. To identify if an interaction with LIN-1 is the source of EGL-38 cell-specificity, we are investigating LIN-1 and its role in the creation of the vulval-uterine connection. We have discovered that a loss-of-function mutation in lin-1 significantly decreases lin-3 expression in the vulF cells, and further experiments are being conducted to examine the in vivo interaction of EGL-38 and LIN-1. Additionally, we are investigating the activation and role of EGL-38 in the uv1 cells to determine how PAX proteins may function discriminately in communicating cells.

References:
1. Rajakumar V and Chamberlin HM. 2007. “The Pax2/5/8 gene egl-38 coordinates organogenesis of the C. elegans egg-laying system.” Developmental Biology 301(1):240-253. PMID: 17020758
2. Chang C, et al. 1999. “Reciprocal EGF signaling back to the uterus from the induced C. elegans vulva coordinates morphogenesis of epithelia.” Current Biology 9(5):237-246. PMID: 10074449
3. Fitzsimmons D, et al. 2001. “Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly.” Nucleic Acids Research 29(20):4154-4165. PMID: 11600704

Keywords: coordinated development, signaling, cell-specificity

50. Genetic Analysis of Retinal Homeobox Enhancer UCE2

Jennifer Bosse (Molecular, Cellular and Developmental Biology, OSU), Reyna Martinez-De Luna (Ophthalmology, SUNY Upstate Medical University)

Abstract not available online - please check the printed booklet.

51. Rapid clearance of oscillating transcripts during somitogenesis requires the decay adapter Pnrc2

Kiel T. Tietz (MCDB), Thomas L. Gallagher (Department of Molecular Genetics, The Ohio State University), Nicolas L. Derr (Department of Molecular Genetics, The Ohio State University), Courtney E. French (Department of Plant and Microbial Biology, University of California, Berkeley), Jasmine M. McCammon (Department of Molecular and Cell Biology, University of California, Berkeley ), Michael L. Goldrich (Department of Molecular and Cell Biology, University of California, Berkeley )

Abstract not available online - please check the printed booklet.

52. A novel role for Scleraxis in promoting valve interstitial cell quiescence

Matt VandeKopple (Molecular, Cellular, and Developmental Biology, The Ohio State University), Joy Lincoln (Center for Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Childrens Hospital)

Abstract:
Heart valve disease affects 2.5% of the general population yet little is known about the
pathophysiology and current therapies remain limited. In healthy valves, function is achieved by
neatly stratified layers of extracellular matrix (ECM) that provide all the necessary
biomechanical properties throughout life. ECM secretion and organization is established during
development and mediated by embryonic activated valve interstitial cells (EaVICs). These
precursor cells mimic myofibroblasts and express high levels of α-smooth muscle actin (SMA).
After birth, EaVICs undergo quiescence, lose SMA expression and serve to maintain valve
homeostasis in the absence of disease. However, in response to pathological stimuli quiescent
VICs become activated and promote pathological ECM remodeling leading to compromised
biomechanical function. Therefore it is imperative that VIC quiescence is maintained to prevent
disease processes. Despite this, regulators of this process have yet to be identified. Here we
use in vivo and in vitro assays to show that the bHLH transcription factor Scleraxis (Scx) is
required for VIC quiescence after birth, as loss of function leads to prolonged expression of
activated VIC markers including SMA and Periostin. Ongoing studies are utilizing a new
transgenic model to determine if Scx is sufficient to prevent VIC activation and valve disease in
a genetically susceptible mouse model. Overall, these studies suggest that Scx is a novel
regulator of VIC phenotypes, thereby providing insights into mechanistic-based therapies in the
prevention and treatment of valve disease.

Keywords: Heart Valve, Scleraxis, Myofibroblast

53. NF-kappaB Function in Neonatal Muscle Development

Jinmo Gu (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University), Denis Guttridge (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

54. Dynamic damage searching and substrate processing by hOGG1 and its coordination with APE1 during base excision repair

Austin T. Raper (Ohio State Biochemistry Program, Department of Chemistry and Biochemistry, The Ohio State University), Brian A. Maxwell (Ohio State Biophysics Graduate Program), Zucai Suo (Ohio State Biochemistry Program, Ohio State Biophysics Graduate Program, Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Base excision repair (BER) is the primary pathway by which our cells resolve single base damage. One particularly common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxo-dG). The ability of 8-oxo-dG to trigger mutation and its high frequency of occurrence warrant its rapid and efficient repair. While the elegant coordination of BER enzymes is implicated in the repair of base lesions, it remains unclear how this coordination is executed in resolving 8-oxo-dG damage. In addition, the mechanism by which our cells limit cytotoxic BER intermediates from eliciting further DNA damage is unclear. Our single-molecule Förster Resonance Energy Transfer (smFRET) technique has provided a means to characterize the enzymes important in the repair of 8-oxo-dG and the potential coordination between them. We specifically explore the interaction of two enzymes involved in consecutive steps in the pathway, human 8-oxoguanine glycosylase (hOGG1) and human AP endonuclease (APE1). In addition to providing valuable insight into the mechanism of hOGG1, our work strongly supports the existence of a ternary complex between hOGG1, APE1, and the DNA substrate. As such, our results imply a mechanism in which APE1 actively displaces hOGG1 from its abasic site product. Furthermore, this work supports the idea that APE1 serves as the connection between the damage specific and general stages of BER by utilizing specific protein-protein interactions with the glycosylase.

Keywords: Single-molecule Frster Resonance Energy Transfer, Base Excision Repair, oxidative damage

55. Kinetic investigation of human polymerases bypassing a pollution-induced DNA lesion

Jack Tokarsky (Biophysics Graduate Program), Varun V. Gadkari (Ohio State Biochemistry Program), Ashis K. Basu (Department of Chemistry, University of Connecticut), Zucai Suo (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

56. Pre-steady-state kinetic characterization of human DNA polymerase ε

Walter J. Zahurancik (The Ohio State Biochemistry Program), Seth J. Klein (Department of Molecular Genetics, The Ohio State University), Zucai Suo (The Ohio State Biochemistry Program)

Abstract:
Maintenance of eukaryotic genome stability requires the action of DNA polymerases (Pols) that are capable of replicating genomic DNA efficiently and accurately. Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to an overall low polymerization error frequency (one error per 10⁶-10⁸ incorporations) and support faithful eukaryotic genome replication. Three replicative DNA polymerases from the B-family, Polα, Polδ, and Polε, are responsible for the majority of eukaryotic DNA replication. Polε has been shown to catalyze leading-strand DNA synthesis during DNA replication in vivo, but little is known about the kinetic mechanism and overall fidelity of polymerization catalyzed by this replicative enzyme. We have utilized pre-steady-state kinetic methods to elucidate the mechanism of DNA polymerization catalyzed by human Polε (hPolε) and provide a kinetic basis for high fidelity DNA replication. Our results show that hPolε follows an induced-fit mechanism by which it exhibits large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides, resulting in a low frequency of mismatched base pair formation (10^-4-10^-7 per nucleotide incorporation event). The 3′→5′ exonuclease activity of hPolε further enhances polymerization fidelity by an unprecedented 3.5x10² to 1.2x10⁴-fold. With an overall fidelity of 10⁶-10⁸ (0.1-1.0 error per round), hPolε is conclusively justified in being the primary enzyme to replicate the human nuclear genome. Consistently, somatic mutations in hPolε, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.

References:
Zahurancik, W. J., Klein, S.J., Suo, Z.* (2014) Significant Contribution of the 3’→5′ Exonuclease Activity to the High Fidelity of Nucleotide Incorporation Catalyzed by Human DNA Polymerase ε. Nucleic Acids Research. 42 (22), 13853-60.

Zahurancik, W.J., Klein, S.J., Suo, Z.* (2013) Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε, Biochemistry. 52, 7041−7049.

Keywords: Pre-steady-state kinetics, Human DNA polymerase epsilon, 35 exonuclease activity

57. Binding Dynamics and Nucleotide Selectivity of Dpo4 in Response to Oxidative Damage as Revealed by Single-Molecule FRET

Varun V. Gadkari (Ohio State Biochemistry Program), Austin T. Raper (Ohio State Biochemistry Program), Brian A. Maxwell (Biophysics), Ahmed S. Beydoun (The Ohio State University, Department of Physics )

Abstract:
Translesion synthesis (TLS) DNA polymerases utilize a complex, multi-step mechanism for efficient bypass of damaged DNA to rescue stalled replication forks as revealed by extensive kinetic and structural studies. Dpo4, the lone Y-family DNA polymerase in S. solfataricus, is likely tasked with TLS responsibilities in response to various lesions in vivo and has been studied extensively as a model Y-family DNA polymerase. Previous stopped-flow fluorescence and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Recently, single-molecule fluorescence spectroscopy has provided new insights into real-time conformational dynamics of DNA polymerases during substrate binding and nucleotide incorporation. We used single-molecule Förster resonance energy transfer (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 during substrate binding. Our findings demonstrate that this TLS polymerase interacts dynamically with the DNA, sampling a variety of locations and conformations as it recognizes and specifically binds the nucleic acid substrate. We observe changes in binding distributions and dissociation kinetics upon the introduction of nucleotide. Furthermore, the overall binding behavior observed on an unmodified DNA substrate is largely conserved in the presence of an oxidative lesion with a few notable caveats including an increase in complex stability and a shift in conformational distribution.

Keywords: Single Molecule FRET, Polymerase, Oxidative Damage

58. Kinetic Investigation into the Mechanism of Pyrococcus furiosus and Thermococcus kodakarensis Trm10

Samantha Dodbele (Ohio State Biochemistry Program, The Center for RNA Biology), Aiswarya Krishnamohan (Ohio State Biochemistry Program, The Center for RNA Biology), Jane E. Jackman (Ohio State Biochemistry Program, The Center for RNA Biology)

Abstract not available online - please check the printed booklet.

59. Deciphering the mechanism of the six-bladed β-propeller hydrolases

Timothy J. Grunkemeyer (OSBP), Dr. Kiran Doddapaneni (Department of Chemistry and Biochemistry, The Ohio State University), Srividya Murali (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Each year approximately 3 million people are exposed to toxic organophosphorous compounds (OPs), 10% of which succumb this exposure. This has led to the search for potential therapeutics in the form of bioscavengers. Several such bioscavengers are the closely related enzymes squid di-isopropylfluorophosphatase (DFPase), bacterial drug responsive protein 35 (Drp35), recombinant Paraoxonase-1 (PON1), and human senescence marker protein 30 (SMP30). These enzymes share the six-bladed β-propeller fold as well as promiscuous substrate specificities. However, due to a plethora of conflicting mechanistic evidence, their enzymatic mechanisms are poorly understood, thus limiting our protein engineering capabilities. In order to clarify the mechanisms, a comparative enzymology approach is being employed with DFPase, Drp35, PON1, and SMP30. We hypothesize that all four enzymes operate via a common mechanism. The wild type (WT) and critical active site mutants have been cloned and the expression and purification protocols have been optimized in a bacterial system. To interrogate their substrate tolerance, each construct has been screened against a panel of lactones, aryl esters, and OPs. All WT enzymes show strong lactonase activity while WT DFPase, PON1, and SMP30 will hydrolyze OPs, albeit with less specificity. In agreement with our hypothesis, mutations of the active site Asp abolish DFPase, PON1, and SMP30 OPase activity. Even though DFPase and SMP30 lose all lactonase activity with the Asp mutation, both PON1 and Drp35 retain minimal activity toward aromatic lactones. Additionally, crystallographic analyses are also under way; strong crystal leads from the Drp35 active site mutants and diffraction patterns of the DFPase active site Asp mutant have been obtained; subsequent structure solution from the latter is in progress. In the future, the mechanism and active site of these enzymes will be interrogated using inhibitor binding and subsequent structural analyses.

Keywords: mechanism, comparative enzymology, structural biology

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

Insiya Fidai (Biophysics Graduate Program), Christine Wachnowsky (Ohio State Biochemistry Program), James A Cowan (Department of Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

61. Investigation of non-pyrrolysine homologs of the pyrrolysine-containing trimethylamine-corrinoid methyltransferase

Jonathan W. Picking (The Ohio State University), Tomislav Ticak (Miami University), Duncan J. Kountz (The Ohio State University), Edward J. Behrman (The Ohio State University), Donald J. Ferguson (Miami University), Joseph A. Krzycki (The Ohio State University)

Abstract not available online - please check the printed booklet.

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

Balasubramani Hariharan (Biophysics), Raunak Soman (OSBP)

Abstract not available online - please check the printed booklet.

63. The role of RCAN1-4 as a tumor metastasis suppressor

Chaojie Wang (Ohio State Biochemistry Program), Adlina Mohd Yusof (Divisions of Endocrinology, Diabetes, and Metabolism, The Ohio State University College of Medicine), Hiroshi Nakanishi (Divisions of Endocrinology, Diabetes, and Metabolism, The Ohio State University College of Medicine), Xiaoli Zhang (Center for Biostatistics, The Ohio State University College of Medicine), Motoyasu Saji (Divisions of Endocrinology, Diabetes, and Metabolism, The Ohio State University College of Medicine), Matthew D. Ringel (Divisions of Endocrinology, Diabetes, and Metabolism, The Ohio State University College of Medicine)

Abstract not available online - please check the printed booklet.

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

Kyle M. LaPak (Molecular, Cellular, and Developmental Biology), Michael Butrey (Arts and Sciences, The Ohio State University), Michael ODwyer (Trinity College Dublin)

Abstract not available online - please check the printed booklet.

65. Stromal PDGFRA activation stalls mammary ductal development and makes the epithelium more tumorigenic

Anisha Mathur (OSBP), Gina Sizemore (OSU-CCC), Vasudha Shukla (Biomedical Engineering), Subhasree Balakrishnan (OSU-CCC), Samir Ghadiali (Biomedical Engineering ), Michael Ostrowski (OSU-CCC)

Abstract not available online - please check the printed booklet.

66. Targetable CREB1/FoxA1-driven prognostic gene expression relies on cyclic nucleotide-dependent kinase-mediated MED1 phosphorylation in prostate cancer

Benjamin Sunkel (Ohio State Biochemistry Program), Dayong Wu, Zhong Chen (Department of Molecular Virology, Immunology, and Medical Genetics, the Ohio State University), Zhenqing Ye (University of Texas Health Science Center at San Antonia), Victor X. Jin (University of Texas Health Science Center at San Antonia), Steven K. Clinton (Department of Internal Medicine, College of Medicine, the Ohio State University), Qianben Wang (Department of Molecular Virology, Immunology, and Medical Genetics, the Ohio State University)

Abstract:
Identifying disease-driving transcription factor networks in addition to the androgen receptor in prostate cancer promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of the master transcription factors (TFs) CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines and primary prostate cancer tissues to investigate their role in defining prostate cancer gene expression profiles. We identified a CREB1/FoxA1 up-regulated 19-member cell cycle-related gene Panel A and a CREB1/FoxA1 repressed 6-member putative tumor suppressor gene Panel B which were prognostic of more rapid prostate cancer recurrence independent of clinical prognosticators in Cox proportional hazard models. We also found that these panels improved upon clinical features in predicting future recurrence events via logistic regression. Further analysis of our 19-gene panel revealed that its expression was potentiated by enhanced CREB1 phosphorylation, and we investigated the utility of inhibiting known CREB1-targeting kinases in disrupting this signaling. Rather than reducing CREB1 phosphorylation levels, cyclic nucleotide-dependent protein kinase inhibition reduced the phosphorylation of an important CREB1 coactivator, MED1, resulting in abrogated target gene expression and cell proliferation. We finally asked how accumulating cyclic nucleotide levels impact this signaling axis, and found that phosphodiesterase inhibitor treatment, commonly utilized in managing erectile dysfunction following radical prostatectomy, significantly enhanced cell cycle gene expression and prostate cancer cell proliferation. In total, our findings establish a central role for CREB1 in driving prostate cancer progression, and demonstrate the utility of CREB1/FoxA1 target gene expression levels as a biomarker for identifying at-risk patients that may benefit from trials of adjuvant treatment. Critically, our work also provides a molecular understanding of the risks associated with phosphodiesterase treatment in the prostate cancer setting, supporting epidemiologic studies of their deleterious effect on treatment outcomes.

Keywords: Prostate cancer, CREB1, PDEi

67. Activation of CSF1 Signaling in Tumor Associated Macrophages during Breast Cancer Brain Metastasis

Katie Thies (Molecular, Cellular and Developmental Biology Graduate Program), Haritha Mathsyaraja (The Comprehensive Cancer Center, The Ohio State University), W. Hans Meisen, Balveen Kaur (Department of Neurological Surgery), Tom Liu, Cynthia Timmers (The Comprehensive Cancer Center, The Ohio State University), Jose Otero (Department of Pathology, The Comprehensive Cancer Center), Michael C. Ostrowski (The Comprehensive Cancer Center, The Ohio State University)

Abstract not available online - please check the printed booklet.

68. Loss of Stromal PTEN induces genomic instability through paracrine EGFR activation

Subhasree Balakrishnan (Department of Molecular Virology, Immunology and Medical Genetics; Solid Tumor Biology Program, Comprehensive Cancer Center), Gina M. Sizemore (Department of Molecular Virology, Immunology and Medical Genetics; Solid Tumor Biology Program, Comprehensive Cancer Center), Anthony J. Trimboli (Department of Molecular Virology, Immunology and Medical Genetics; Solid Tumor Biology Program, Comprehensive Cancer Center), Anisha Mathur (Department of Molecular Virology, Immunology and Medical Genetics; Solid Tumor Biology Program, Comprehensive Cancer Center), Raleigh Kladney (Solid Tumor Biology Program, Comprehensive Cancer Center), Dongju Park (Department of Molecular and Cellular Biochemistry, College of Medicine; Solid Tumor Biology Program, Comprehensive Cancer Center)

Abstract not available online - please check the printed booklet.

69. Inhibition of Liver Tumorigenesis by Blocking CCL2-CCR2 Axis, A Target of MIR-122

Kun-Yu Teng (MCDB), Jianfeng Han, Michael ACaliguiri (Department of Internal Medicine, CCC, OSU), Shu-Hao Hsu (Department of Pathology, CCC, OSU), Samson Jacob (Molecular & Cellular Biochemistry, CCC, OSU), Jianhua Yu (Department of Internal Medicine, CCC, OSU), Kalpana Ghoshal (Department of Pathology, CCC, OSU)

Abstract not available online - please check the printed booklet.

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

Mohamad Elbaz (OSU), Mohd Nasser (osu), janani Ravi (OSU), Nissar wani (OSU), Grace Amponsah (OSU), Ramesh Ganju (OSU)

Abstract not available online - please check the printed booklet.

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

Pooja Joshi, Young-Jun Jeon (MCDB), Alessandro Lagana (Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY), Justin Middleton (MCDB), Paola Secchiero (Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, Italy), Michela Garofalo (Transcriptional Networks in Lung Cancer Group, Cancer Research UK Manchester Institute, University of Manchester, UK), Carlo M. Croce (Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University)

Abstract not available online - please check the printed booklet.

72. Targeting CDKs with PHA-848125 in estrogen receptor negative breast cancer

Douglas G. Cheung (Molecular, Cellular, and Developmental Biology 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.

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

Yi Seok Chang (Department of Molecular and Cellular Biochemistry), Swati P. Jalgaonkar (Department of Molecular and Cellular Biochemistry), Seung Ho Jung (Institute for Behavioral Medicine Research), John Sheridan (Institute for Behavioral Medicine Research), Tsonwin Hai (Department of Molecular and Cellular Biochemistry)

Abstract not available online - please check the printed booklet.

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

Swati P. Jalgaonkar (MCDB), Yi Seok Chang (MCDB), Justin Middleton (MCDB), Christopher Wolford (MCB), Bhuvaneswari Ramaswamy (Internal Medicine), Tsonwin Hai (MCB)

Abstract not available online - please check the printed booklet.

75. The role of PALB2 in BRCA1-mediated DNA repair and tumor suppression.

Dongju Park (Molecular, Virology, Immunology & Medical Genetics), Reena Shakya (Comprehensive Cancer Center), Jose A. Colina (Ohio State Biochemistry Program), Alejandra M. Maiz (Ohio State Molecular Genetics Program), Ashley Hajis (Ohio State Biology Program), Harvey Lewis (Ohio State Biochemistry Program)

Abstract not available online - please check the printed booklet.

76. Genome-wide effects on alternative splicing networks by dietary flavonoids restore normal RNA-signatures contributing to halt breast cancer progression.

Daniel Arango (Molecular Genetics. Physiology and Cell Biology. Heart and Lung Research Institute), Katherine Mejia-Guerra (Molecular Genetics. Center for Applied Plant Sciences.), Eric Mukundi, Francisco Padilla-Obregon (Center for Applied Plant Sciences.), Timothy D. Eubank (Heart and Lung Research Institute), Erich Grotewold (Molecular Genetics. Center for Applied Plant Sciences.), Andrea I. Doseff (Molecular Genetics. Physiology and Cell Biology. Heart and Lung Research Institute)

Abstract not available online - please check the printed booklet.

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

Tasha Wilkie (MCDB)

Abstract not available online - please check the printed booklet.

78. Interferon gamma promotes natural killer cell-specific immunoevasive strategies in multiple myeloma

Paul David Ciarlariello (Molecular Cellular and Developmental Biology Graduate Program, OSU), Daniel M Banaszak (Biomedical Science Major Undergraduate Program, OSU), Tiffany Hughes, PhD (Division of Hematology Oncology, OSU-CCC), Courtney E Bakan (Division of Hematology Oncology, OSU-CCC)

Abstract not available online - please check the printed booklet.

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

Kaitlin Snider (Department of Neuroscience, Ohio State University), Katelin Hansen, Heather Dziema, Jacob Loeser, Sydney Aten (Department of Neuroscience, Ohio State University), Ian Sanchez (Postbaccalaureate Research Education Program, Ohio State University), Carl Pelz (Department of Cell and Developmental Biology, Oregon Health and Science University), Soren Impey (Department of Cell and Developmental Biology, Oregon Health and Science University), Karl Obrietan (Department of Neuroscience, Ohio State University)

Abstract not available online - please check the printed booklet.

80. Altered immune signaling impairs oligodendrocyte lineage cell responses and functional recovery after spinal cord injury in mice

Jamie Church (Neuroscience Graduate Program), Phillip Popovich (Neuroscience, Ohio State University), Dana McTigue (Neuroscience, Ohio State University)

Abstract:
Prominent oligodendrocyte (OL) loss occurs after spinal cord injury (SCI), followed by a rebound in the first 2 weeks post-injury due to proliferation and differentiation of surviving OL progenitor cells (OPCs). The mechanisms controlling this endogenous response, however, are not understood. Previous work from our group showed activation of toll-like receptor 4 (TLR4) on microglia induced OPC proliferation and oligodendrogenesis in the intact spinal cord. Since TLR4 ligands are present in the injured spinal cord, we tested the hypothesis that TLR4 signaling contributes to oligodendrogenesis after SCI. Wild-type (WT) and TLR4-deficient (TLR4d) mice were given a midthoracic moderate contusion SCI and sacrificed for spinal cord histology and PCR at 1, 3, 7, 14, or 21d post-injury; behavioral analyses and axon and myelin integrity analyses were conducted on a separate set of animals surviving up to 42d. Reduced locomotor recovery in TLR4d mice coincided with reduced OL lineage cell responses and less spared tissue chronically in ventral motor tract areas. TLR4d mice had increased OL loss acutely concurrent with decreased ferritin (iron storage) expression in the lesion, suggesting TLR4 signaling helps protect against acute iron-mediated damage. TLR4d mice had reduced OL numbers and OPC markers chronically despite enhanced acute OPC proliferation. Inhibitors of OL differentiation were increased in TLR4d mice, likely promoting lower chronic OL numbers. Additionally, delayed lipid accumulation (debris phagocytosis) in TLR4d mice could contribute to reduced OPC and OL responses. Interestingly, growth factor expression was elevated, suggesting that TLR4 signaling normally suppresses growth factor expression after SCI. Collectively, this work shows that post-SCI OL protection and replacement are influenced by the inflammatory environment, and highlights the importance of TLR4 signaling for normal cellular repair mechanisms, growth factor expression, and functional recovery.

Keywords: TLR4, Spinal cord injury, oligodendrocyte

81. The Role of hsa-miR-30c-5p in Glioblastoma Multiforme

Theresa Relation (Neuroscience Graduate Program, Medical Scientist Training Program), Ji Young Yoo (Department of Neurological Surgery), Chelsea Bolyard (Department of Neurological Surgery), Selene Virk (Department of Epidemiology and Statistics, Case Western Reserve University), Jill Barnholtz-Sloan (Department of Epidemiology and Statistics, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

82. Social Influences on Neuroinflammation after Ischemia

Monica M. Gaudier-Diaz (Neuroscience Graduate Program), Ning Zhang (Department of Neuroscience), Min Zhou (Department of Neuroscience), Courtney DeVries (Department of Neuroscience)

Abstract not available online - please check the printed booklet.

83. Microglial Alterations Within the Postpartum Brain

Achikam Haim (Neuroscience, The Ohio State University ), Kathryn M Lenz (Psychology and Neuroscience, The Ohio State University ), Benedetta Leuner (Psychology and Neuroscience, The Ohio State University)

Abstract:
The postpartum brain is remarkably plastic. In numerous brain regions, motherhood is associated with alterations in the production of new neurons as well as neuronal dendritic and synaptic remodeling. Postpartum modifications are not limited to neurons however, as astrocytic changes have also been documented. Possible postpartum-related alterations in microglia, the primary innate immune cells of the brain, hasn’t been examined although such a consequence is likely given the well-known peripheral immune changes that occur at this time, the role that microglia play in regulating neuronal plasticity, and the sensitivity of microglia to various hormones that are altered postpartum. To begin investigating this possibility, coronal brain sections from postpartum day 8 and nulliparous rats were stained for Iba1, a pan-microglial marker. Densitometry of Iba1 staining was performed in the CA1, CA3, and dentate gyrus subfields of the hippocampus. Our results show that compared to nulliparous females, Iba1 density was reduced in all three subfields of the postpartum hippocampus. Although preliminary, these results are the first to reveal microglial alterations within the postpartum brain. We are currently investigating whether reduced Iba1 density reflects a reduction in microglia number or a change in microglial phenotype as well as the timing of these changes, their regional specificity, and the underlying mechanism. Together, these studies will uncover whether microglia are regulated by the experience of motherhood and will lead to future studies investigating whether microglia, in turn, regulate the brain and behavioral changes that occur postpartum. The role microglia play in developmental plasticity is well known, and the postpartum period may provide a unique opportunity to gain a better understanding of how microglia modulate plasticity within the healthy adult brain.

Keywords: Postpartum , Microglia , Hippocampus

84. Improved Gene Delivery to Adult Mouse Spinal Cord Through the Use of Hybrid Adeno-Associated Viral (AAV) Vectors

Jason J. Siu (Medical Scientist Training Program, Neuroscience Graduate Studies Program), Chuansong Wang (Department of Molecular Virology, Immunology, and Medical Genetics), Lei Cao (Department of Molecular Virology, Immunology, and Medical Genetics)

Abstract not available online - please check the printed booklet.

85. Sex differences in the response to exposure to light at night and high fat diet during early life

Yasmine M. Cisse (Department of Neuroscience and Neurological Institute, The Ohio State University Wexner Medical Center), Randy J. Nelson (Department of Neuroscience and Neurological Institute, The Ohio State University Wexner Medical Center)

Abstract not available online - please check the printed booklet.

86. Hedgehog signaling in the formation of Muller glia-derived progenitor cells.

Levi Todd (Department of Neuroscience), Andy Fischer (Department of Neuroscience)

Abstract:
The capacity of retinal regeneration varies substantially across vertebrate species, but consistently involves Müller glia as the cellular source for progenitors. Although retinal regeneration is robust in fish, the regeneration in birds and mammals is limited. However, after retinal injury, Müller glia in the avian retina are able to undergo a program of de-differentiation, proliferation, and neurogenesis. The signaling pathways that influence the formation of Müller glia-derived progenitor cells (MGPCs) are slowly being revealed. The purpose of this study was to investigate whether Hedgehog-signaling is a key node in the signaling network that orchestrates the regenerative response of MGPCs. In damaged retinas, we found that Hedgehog-signaling is dynamically up-regulated in Müller glia/MGPCs, and inhibition of Hedgehog-signaling decreases numbers of MGPCs. By comparison, activation of Hedgehog-signaling increases the formation of MGPCS in moderately damaged retinas, but is not sufficient to induce the formation of MGPCs in the absence of injury. Fibroblast growth factor 2 (FGF2) alone induces the formation of MGPCs in undamaged retina. We found that Hedgehog-signaling is necessary for FGF2-induced MGPC formation as Hedgehog inhibition prevents progenitor cell formation. Additionally, co-application of Hedgheog agonists with FGF2 leads to an increase of MGPC formation. We conclude that Hedgehog-signaling stimulates the formation of proliferating MGPCs in acutely damaged retinas. Further, in undamaged retinas, our data indicate that FGF2/MAPK-signaling recruits Hedgehog-signaling to boost the regenerative response of MGPCs.

Keywords: retina, glia, regeneration

87. Cytotoxic chemotherapy increases sleep and sleep fragmentation in non-tumor-bearing mice

Jeremy C Borniger (Neuroscience Graduate Program), Monica M Gaudier-Diaz (Neuroscience Graduate Program), Ning Zhang (Department of Neuroscience), Randy J Nelson (Department of Neuroscience), A Courtney DeVries (Department of Neuroscience)

Abstract not available online - please check the printed booklet.

88. Protocadherins control the modular assembly of neuronal columns in the zebrafish optic tectum

Sharon R. Cooper (MCDB), Michelle R. Emond (Department of Neuroscience, Ohio State University), Phan Q. Duy (Department of Neuroscience, Ohio State University), Brandon G. Liebau (Department of Neuroscience, Ohio State University), Marc A. Wolman (Department of Zoology, University of Wisconsin), James D. Jontes (Department of Neuroscience, Ohio State University)

Abstract not available online - please check the printed booklet.

89. Spinal cord injury triggers synthesis of autoantibodies against axonal protein collapsin response mediator protein 2 (CRMP2)

Jessica M. Marbourg (Department of Neuroscience, The Ohio State University), Yi Zhang (Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University ), Dan P. Ankeny (Department of Molecular Virology, Immunology & Medical Genetics, The Ohio State University), Wenmin Lai (Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University), Jan M. Schwab (Department of Neurology and Experimental Neurology, Charite-Universitatsmedizin Berlin, Germany), Phillip G. Popovich (Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University)

Abstract not available online - please check the printed booklet.

90. Human immune cells infiltrate the lesion of humanized mice after contusive spinal cord injury

Randall S. Carpenter (Neuroscience Graduate Program, The Ohio State University, Columbus, OH), Kristina A. Kigerl (Center for Brain and Spinal Cord Repair, The Ohio State University, Columbus, OH), Stefan Niewiesk (Department of Veterinary Biosciences, 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:
Mice are important pre-clinical models for investigating inflammation and immune system function after acute spinal cord injury (SCI). However, recent data indicate that the composition and function of mouse and human immune systems differ. Understanding the human immune response to SCI and the development of novel immune modulatory therapies may benefit from the generation of animal models that better reflect the human condition. Humanized mice are immunocompromised mice engrafted with human blood stem cells, resulting in the development of a human immune system. The purpose of the current study was to determine if humanized mice can be used to investigate human immune responses to acute SCI. Newborn NOD-SCID-IL2rg null (NSG) mice underwent mild irradiation followed by injection of 50K human CD34+ hematopoietic stem cells into the liver. Ten weeks after engraftment human immune cells made up 8% of all peripheral white blood cells in humanized NSG (hNSG) mice. Thirteen weeks after engraftment hNSG mice were subjected to a moderate thoracic spinal cord contusion injury. After SCI, the proportion of human immune cells increased significantly to 57%, likely due post-injury changes in hematopoiesis. Also, human T cells, B cells, and macrophages were identified in peripheral organs. Human immune cells infiltrated the spinal cord by 28 dpi and were confined to the lesion core, internal to the glial scar. The composition of human immune cells in the lesion included human macrophages, T cells, and B cells. Human macrophages displayed a large, phagocytic phenotype and were the most abundant human immune cell in the lesion. Human T cells were interspersed among phagocytic macrophages, occasionally forming small clusters within and surrounding the lesion. Human B cells in the lesion were rare, and could only be identified in a subset of hNSG mice. These results indicate that humanized mouse models can be utilized to investigate human neuroinflammation after SCI.

Keywords: Neurotrauma, Humanized Mice, Neuroimmunology

91. Interactions between corticospinal and reticulospinal outputs determine muscle response in the upper limbs and trunk

Sarah M. Hulbert (Biophysics Graduate Program), John A. Buford (Division of Physical Therapy, College of Medicine, The Ohio State University)

Abstract:
It is known that both corticospinal and reticulospinal tracts individually contribute to the stability and movement of the upper limbs and trunk. Using stimulus-triggered averaging (StimTA) of motor cortex and pontomedullary reticular formation (PMFR) outputs, we tested the hypothesis that these regions also interact to produce effects (suppression or facilitation) in upper limbs and trunk. In our paired pulse paradigm, three areas of the cortex-primary motor cortex (M1), supplementary motor area (SMA), and dorsal premotor cortex (PMd)-were electrically stimulated at varying time shifts with respect to the electrical stimulation of the PMRF during a bilateral reaching task. Specifically, three paradigms were used: 1) Cortical area stimulated before PMRF, 2) Both areas stimulated simultaneously, 3) The PMRF stimulated before the cortical area. These experiments were conducted in three non-human primates, M. fascicularis .
We studied 12 bilateral pairs of muscles in the upper limbs and trunk. By comparing EMG results from the individual stimulation of the cortex or the PMRF to EMG results from the paired pulse paradigms, we were able to identify muscle responses dominated by a single region (either cortex or PMRF) as well as responses indicative of complex interactions between the regions.
As expected, we were able to detect several instances where stimulation of a single region, usually the PMRF, resulted in a significant muscle response and that same region was shown to be responsible for the muscle responses in each of the paired pulse paradigms. One of the most prominently observed complex interactions was the gating of the cortex by the PMRF. That is, individual stimulation of the cortex revealed a significant muscle response, as did individual stimulation of the PMRF. However, in at least one of the paired pulse paradigms, the muscle response was significantly attributed to the PMRF only, indicating that signals from the PMRF negated signals coming from the cortical areas.
This shows that corticospinal and reticulospinal systems can interact to produce muscle recruitment. Therefore, a systems approach is needed to fully understand the outputs of these motor systems.

Keywords: Pontomedullary reticular formation, motor cortex, reaching, stimulus-triggered averaging

92. AAV9-HSPB1: a novel tool for accelerating motor unit repair following peripheral nerve injury

Christopher G. Wier (Neuroscience), Lisa N. Miller (Biomedical Engineering, OSU), Patrick L. Heilman (Molecular & Cellular Biochemistry, OSU), Xueqian Wang (Molecular & Cellular Biochemistry, OSU), W. David Arnold (Neurology, Physical Medicine and Rehabilitation, Neuroscience, OSU), Stephen J. Kolb (Neurology, Molecular & Cellular Biochemistry, OSU)

Abstract:
The motor unit is made up of a motor neuron and all muscle fibers that receive its innervation. Improving motor unit repair following a denervating injury or disease is a crucial research area to ensure return of muscle function. The small heat shock protein B1 (HspB1) is a promising candidate for accelerating motor unit repair and recovering muscle function. HSPB1 expression increases in motor neurons and glial cells following neuronal injury and transgenic lines overexpressing the protein result in accelerated axonal growth [1-3]. We have built off our prior work involving self-complementary adeno-associated virus serotype 9 (scAAV9) to develop a tool for targeting HSPB1 overexpression in motor neurons and astrocytes, AAV9-HSPB1. A longitudinal motor unit repair time course has been produced in mice with a sciatic nerve crush using a combination of compound muscle action potential (CMAP) and motor unit number estimate (MUNE) measurements—recordings performed by clinicians on patients with peripheral nerve injury—and paw print measurements. We have demonstrated accelerated motor unit repair in mice injected with AAV9-HSPB1 compared to non-injected mice, with CMAP and MUNE returning to pre-crush levels by 42 days post injury (dpi) versus approximately 80dpi.

References:
1. Costigan, M., et al., Heat shock protein 27: developmental regulation and expression after peripheral nerve injury. J Neurosci, 1998. 18(15): p. 5891-900.
2. Benn, S.C., et al., Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron, 2002. 36(1): p. 45-56.
3. Ma, C.H., et al., Accelerating axonal growth promotes motor recovery after peripheral nerve injury in mice. J Clin Invest, 2011. 121(11): p. 4332-47.

Keywords: scAAV9, HSPB1, Motor unit repair

93. Consequences of Axonal Neuropathy Associated HSPB1 Mutations on NF-kB Stress Signaling Paithways

Patrick L. Heilman (Ohio State Biochemistry Program), Stephen J. Kolb MD., PhD (Molecular and Cellular Biochemistry, OSU.)

Abstract:
Neuroinflammation plays a key role in the pathogenesis of many diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s Disease (AD) and Multiple Sclerosis. One of the major signaling pathways of the neuroinflammatory response is the Nuclear Factor Kappa Beta (NF-κβ) pathway, which regulates the expression of pro and anti-inflammatory cytokines. The small heat shock protein 27 (HSPB1) is a molecular chaperone that displays neuroprotective properties in many disease and injury models [1, 2]. Mutations in HSPB1 result in a late-onset distal axonal neuropathy (dHMN) [3, 4]. HSPB1 regulates the activation of NF-κβ signaling by interacting with multiple upstream protein complexes. Here, we investigated how dHMN causing mutations in HSPB1 affect the NF- κβ pathway. In HeLa cells, overexpression of HSPB1 reduces TNFα-mediated NF-κβ by binding IKKβ and downregulating its activity [5]. We confirmed these results in our lab, and have found that a dHMN-associated mutation in HSPB1 is defective in its ability to alter NF-κβ signaling.

References:
1. Read, D.E. and A.M. Gorman, Heat shock protein 27 in neuronal survival and neurite outgrowth. Biochem Biophys Res Commun, 2009. 382(1): p. 6-8.
2. Sharp, P., et al., Heat shock protein 27 rescues motor neurons following nerve injury and preserves muscle function. Exp Neurol, 2006. 198(2): p. 511-8.
3. Evgrafov, O.V., et al., Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet, 2004. 36(6): p. 602-6.
4. Houlden, H., et al., Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology, 2008. 71(21): p. 1660-8.
5. Park, K.J., R.B. Gaynor, and Y.T. Kwak, Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation. J Biol Chem, 2003. 278(37): p. 35272-8.

Keywords: HSPB1, Axonal Neuropathy, NF-kB

94. Abnormal sensorimotor recovery in a transgenic mouse model of spinal cord injury

Timothy D. Faw, PT, DPT, NCS (Neuroscience Graduate Program, The Ohio State University), Jessica K. Lerch, PhD (Department of Neuroscience, The Ohio State University), Samantha D. Kerr (School of Health and Rehabilitation Sciences, The Ohio State University), Rochelle J. Deibert (School of Health and Rehabilitation Sciences, The Ohio State University), Lesley C. Fisher (School of Health and Rehabilitation Sciences, The Ohio State University), D. Michele Basso, PT, Ed.D. (School of Health and Rehabilitation Sciences, The Ohio State University)

Abstract:
Neuropathic pain is a devastating and often intractable consequence of spinal cord injury (SCI) in up to two-thirds of patients with many reporting a negative impact on quality of life (QOL). Neuropathic pain can manifest as pain from normally non-painful stimuli (allodynia) or heightened response to a noxious stimulus (hyperalgesia). In animal models, these symptoms historically track together, preventing mechanistic study. Here we report atypical sensorimotor recovery after SCI in a Thy1-GFP/M mouse line that, for the first time, allows differential study of allodynia and hyperalgesia. To fully characterize the behavioral phenotype, we performed a severe SCI contusion (SCI) or transection (TX) at T9 in Thy1 GFP+ (GFP+) and Thy1 GFP- (GFP-) littermates. Sensory recovery was examined using the Hargreaves Method and Von Frey Hair Test prior to and 42 days post injury (dpi). Motor recovery was evaluated in the open field using the Basso Mouse Scale prior to injury, at 1 dpi, and weekly thereafter until 42dpi. Locomotor performance compared to historic wild-type (WT SCI) was increased by 7dpi in Thy1 SCI animals (WT SCI = 2.4 +/- 0.7, GFP+ SCI = 4.8 +/- 0.5, GFP- SCI = 5.0 +/- 0.0; GFP+ TX = 0.8 +/- 0.4; p<.01) and remained increased at 42dpi (WT SCI = 5.6 +/- 0.4, GFP+ SCI= 6.5 +/- 0.3, GFP- SCI = 7.5 +/- 0.0; GFP+ TX = 1.3 +/- 0.4; p<.01). Thermal sensitivity increased following SCI and TX in all animals (GFP+ SCI Pre = 9.2sec +/- 1.0, Post = 5.5sec +/- 0.7; GFP- SCI Pre = 7.6sec +/- 2.0, Post = 2.3sec +/- 0.5; GFP TX Pre = 7.9sec +/- 0.8, Post = 4.0sec +/- 0.3; p<.001) indicating hyperalgesia. Unlike WT SCI mice, hypoalgesia occurred in the Thy1-GFP/M line (GFP+ SCI Pre = 0.37g +/- 0.06, Post = 0.89g +/- 0.12;GFP- SCI Pre = 0.75g +/− 0.10, Post = 2.18 +/- 0.61; GFP+ TX; Pre = 0.67g +/- 0.17, Post = 4.33g +/- 0.33; p<.001). The degree of hypoalgesia was different between groups at 42dpi (p<.01). Current experiments are underway to examine white matter sparing at the epicenter and coordination assessed by grid walk. Together, these findings demonstrate a novel model of neuropathic pain where clinically relevant SCI results in hypersensitivity to pain yet hyposensitivity to touch. This divergent profile allows a unique opportunity to examine mechanisms underlying both allodynia and hyperalgesia, which are essential to improving QOL for people with SCI.

References:
1. Detloff et al (2008) Exp Neurol 212(2):337–347. 2. Hansen et al (2013) J Neurosci 33(32):13101–13111. 3. Hoschouer et al (2009) Exp Neurol 216(1):22-34. 4. Hoschouer (2010) PAIN 148(2):328–342. 5. Detloff et al (2010) Exp Neurol 225(2): 366-376.

Keywords: Spinal Cord Injury, Neuropathic Pain, Recovery

95. Development of Small Molecule Translational Activators of Glutamate Transporter, EAAT2, for Treatment of ALS

Joshua Foster (Department of Neuroscience), Sky Dominguez (Department of Neuroscience), Kou Takahashi (Department of Neuroscience), Chien-Liang Glenn Lin (Department of Neuroscience)

Abstract not available online - please check the printed booklet.

96. N-terminal and C-terminal SMN missense mutations complement each other resulting in complete rescue of survival and electrophysiological deficits in Spinal Muscular Atrophy (SMA) mice indicating that snRNP assembly is the key function of SMN in SMA

Chitra C. Iyer (Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus OH, USA), Aurelie Massoni-Laporte, Sandra I. Duque, Narasimhan Madbusi, Thanh T. Le (Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus OH, USA), W. David Arnold (Department of Neurology, The Ohio State University, Columbus OH, USA), Vicki L. McGovern, Eileen Workman, Daniel J. Battle (Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus OH, USA), Sarah Tisdale, Livio Pellizzoni (Center for Motor Neuron Biology and Disease, Columbia University, New York, USA), Arthur H.M. Burghes (Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus OH, USA; Department of Neurology, The Ohio State University, Columbus OH, USA; Department of Molecular Genetics )

Abstract not available online - please check the printed booklet.

97. Identification of Modifiers of Spinal Muscular Atrophy

Corey Ruhno (The Ohio State University), Vicki McGovern (The Ohio State University), Jesse Hunter (TGen), Lisa Baumbach-Reardon (TGen), John Carpten (TGen), Arthur Burghes (The Ohio State University)

Abstract not available online - please check the printed booklet.

98. Identifying critical signals from antigen-presenting cells for generating a pathogenic T cells in multiple sclerosis

Priscilla Lee (Molecular Cellular and Developmental Biology Graduate Program), Yuhong Yang (Department of Neurology), Michael Racke (Department of Neurology), Amy Lovett-Racke (Department of Microbial Infection and Immunity)

Abstract:
While it is generally accepted that Multiple Sclerosis (MS) is a T cell-mediated disease, the factors determining the encephalitogenicity of T cells remains unclear. Myelin-specific T cells generated with antigen presenting cells (APCs) plus myelin peptide are encephalitogenic, whereas T cells generated with anti-CD3/CD28 antibodies are not, suggessting that APCs provide critical signals beyond T cell receptor activation and co-stimulation, contributing to an encephalitogenic phenotype. To recapitulate the signals provided by APCs, naïve myelin-specific CD4+ T cells were activated with anti-CD3/CD28 in the presence of various cytokines. The degree of encephalitogenicity of T cells was examined by the severity of experimental autoimmune encephalomyelitis (EAE). Although no single cytokine was sufficient, the combination of IL-6/IL-23 or IL-12/IL-23 generated highly encephalitogenic T cells. IL-6 and IL-12 induced the initial expression of IL-23R on naïve T cells, and IL-23 further enhanced the expression of its own receptor and the encephalitogenicity. Neutralizing IL-6 or IL-12 with antibodies significantly reduced the severity of EAE, which was induced by APC-generated T cells. Furthermore, IL-23 signling activated not only STAT3, an established pathway under IL-23R, but also STAT4, which was thought to be restricted to Th1 cells. Loss of STAT4 in T cells generated with IL-6/IL-23 ameliorated the severity of EAE, indicating STAT4 is essential for IL-23-induced encephalitogenicity. We have identified that IL-6/IL-23 or IL-12/IL-23 combination in addition to TCR activation and co-stimulation were the miminal signals necessary to generate encephalitogenic T cells. This provides potential targets that can be manipulated therapeutically, resulting in innovative treatments for MS.

Keywords: T cell , multiple sclerosis, autoimmunity

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

Stephanie Hicks (OSBP), Supranee Chaiwatpongsakorn (The Research Institute at Nationwide Childrens Vaccines and Immunity), Mark E. Peeples (The Research Institute at Nationwide Childrens Vaccines and Immunity)

Abstract:
Despite high morbidity and mortality, no vaccine is available for protection against RSV disease. The 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 conformation was recently co-crystalized with monoclonal antibody (mAb) D25 that specifically recognizes the prefusion form and is more highly neutralizing than mAbs that do not bind to the apex of the F protein. The ability of D25 to stabilize the prefusion form and its high neutralizing activity suggests its apical binding site is important for protein function. We hypothesize that residues in this binding site, including those in the apical F2 loop, are required for F protein triggering and, therefore, its function. Twelve residues in the apical loop of the F2 domain were mutated to assess their importance in fusion. Using a mAb that recognizes both protein conformations, it was determined these mutants were expressed on the cell surface. The ability of these mutants to cause fusion was determined with a cell-cell fusion assay. Six of the twelve alanine-scanning mutants were deficient in their ability to cause fusion, five of which were loss of charge mutants. Conservative mutations that retain the charges were able to cause cell-cell fusion, while non-conservative, opposite charge mutations were not. These results suggest the natural charge of each residue, and of the F2 apical loop, in general, is required for F protein function. Each mutant’s cell surface conformation was determined using mAbs specific to the prefusion or postfusion form. Despite an inability to function, most mutants reached the cell surface in the prefusion conformation, indicating premature triggering is not the reason for their loss of function.

Keywords: RSV , Fusion Protein

100. Examination of Human Lysyl-tRNA Synthetase/tRNALys Primer Recruitment and Packaging into HIV-1

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

Abstract:
The primer for reverse transcription in HIV-1, human tRNA^Lys3, is selectively packaged into virions along with tRNA^Lys1,2. Human lysyl-tRNA synthetase (LysRS) , the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNA^Lys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors is required for optimal viral infectivity. LysRS is part of a dynamic mammalian multisynthetase complex (MSC) and has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways. While some aspects of tRNA primer packaging into HIV-1 particles are now understood, the mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into viral particles is unclear. Here, we show that the expression of LysRS is unaltered upon HIV-1 infection, suggesting that the LysRS species packaged is recruited from an existing pool of LysRS. Using immunofluorescence and confocal microscopy with both HEK293T and HuT/CCR5+ cells, we find that LysRS trafficking is altered upon HIV-1 infection with more LysRS localized to the nucleus. LysRS-Gag and LysRS-genomic RNA co-localization studies are currently underway. Our studies also indicate that HIV infection results in phosphorylation of LysRS on Ser/Thr. We hypothesize that LysRS phosphorylation results in release from the MSC and nuclear entry. Studies to understand the significance of these findings for HIV infectivity are in progress.

Keywords: HIV, LysRS, tRNA

101. The Role of Tax-1 and the Alternative NF-kB and Akt Signaling Pathways in HTLV Transformation

Jacob Al-Saleem (Center for Retrovirus Research, The Ohio State University, Columbus, OH, USA), Mathew Cherian (Division of Oncology, Washington University, St Louis, MO, USA), Hicham Baydoun (Division of Oncology, Washington University, St Louis, MO, USA), Mamuka Kvaratskhelia (Center for Retrovirus Research, 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, The Ohio State University, Columbus, OH, USA)

Abstract:
Human T-cell Leukemia Virus Type-1 (HTLV-1) is a complex retrovirus infecting 15-25 million people worldwide, and is the etiological agent of a malignancy of CD4+ T cells termed Adult T-Cell Leukemia. By contrast, HTLV-2 is non-pathogenic in humans. Both HTLV-1 and HTLV-2 express related Tax proteins termed Tax-1 and Tax-2, respectively. Studies have revealed that Tax-1 contains a C-terminal PDZ (post synaptic density protein) domain binding motif (PBM) and a central leucine zipper region (LZR), which are absent in Tax-2. Previous studies indicated that these two domains are important for the ability of Tax-1 to activate alternative the NF-kB signaling pathway. Since Tax-2 is incapable of activating alternative NF-kB signaling we proposed that Tax-1 activation of the alternative NF-kB pathway is important for the HTLV-1 pathogenesis. We set out to identify binding partners of Tax-1 that are important for activation of alternative NF-kB. Using Tax-1 mutants that do not possess the PBM or LZR we identified several potential candidates via a proteomic screen. We plan to utilize siRNA knockdowns to screen these candidates for importance in Tax-1 driven alternative NF-kB activation. During our analyses we found that deletion of the PBM from Tax-1 did not cause a deficiency, but resulted in an enhancement of alternative NF-kB activation. With further analysis, we found that Tax-1 PBM is important for the ability of Tax-1 to activate Akt. Tax-1 diminishes the function of PTEN (Phospatase and Tensin homologue), which inhibits the PI3K-Akt-mTOR pathway. We found that Tax-1, but not PBM deleted Tax-1, competes with PTEN for binding to DLG-1 (Drosophila disk large tumor suppressor), which leads to an increase in Akt activation. These studies suggest that alternative NF-kB and Akt signaling pathways may explain the differences in HTLV-1 and HTLV-2 pathogenesis. Moreover, these findings suggest a new approach to therapeutics for HTLV-1 diseases.

Keywords: HTLV, Akt, NF-kB

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

Tami Coursey (MCDB, Ohio State University), Milica Milutinovic, Jelena Brkljacic (ABRC, Ohio State University), David Bisaro (MCDB, Ohio State University)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

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

Kruthika Sundaram (Biophysics graduate program, The Ohio State University), Mohd. Akhlakur Rahman (Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University), Mark Wewers (Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University)

Abstract not available online - please check the printed booklet.

105. Inflammatory links between air pollution and metabolic syndromes

Cuiqing Liu (Davis Heart and Lung Research Institute), Yuntao Bai (MCDB), Qinghua Sun (Davis Heart and Lung Research Institute), Sanjay Rajagopalan (Davis Heart and Lung Research Institute)

Abstract:
Epidemiological studies have provided a link between air pollution particulate matters (PM) and metabolic disorders. CC-chemokine receptor 2 (CCR2) plays a crucial role in macrophage infiltration into obese adipose tissues. In this study, we aimed to investigate the effects of CCR2 signaling on PM-mediated insulin resistance. C57BL/6 and CCR2-/- mice were fed high-fat diet and exposed to concentrated ambient PM for 17 weeks via Versatile Aerosol Concentrator and Enrichment System. Glucose homeostasis was evaluated via glucose tolerance test and insulin tolerance test; inflammatory cells within visceral adipose tissue were counted using flow cytometry; and insulin signaling was detected via western blot. Our results showed that PM exposure caused insulin resistance, upregulated p38 expression, and recruited more macrophages within adipose tissue in obese wild type mice. These effects were attenuated in CCR2-/- mice. Our date indicate that PM exposure exaggerated the effects of high-fat diet on metabolic disorders; CCR2 may play an important role in PM-mediated insulin resistance through recruiting macrophages into adipose tissues.

References:
Rajagopalan S. Air pollution and type 2 diabetes: mechanistic insights. Diabetes. 2012;61(12):3037-3045.
Shoelson SE. Inflammation and insulin resistance. J Clin Invest. 2006;116(7):1793-1801.
Tsou CL. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J Clin Invest. 2007;117(4):902-909
Bai Y. Macrophage recruitment in obese adipose tissue. Obes Rev. 2015 Feb;16(2):127-36.

Keywords: Air pollution particulate matters, Insulin resistance, CCR2

106. Catalytic hydrogen production in artificial metalloenzymes

Jeffrey W. Slater (The Ohio State University), Haleigh A. Monaco (The Ohio State University), Sabrina L. Cirino (The Ohio State University)

Abstract:
The energy crisis of the last decade has opened the door for creative approaches to solve this urgent problem. Hydrogen has emerged as a leading candidate for an alternative fuel source. Towards this end, hydrogenases, which are enzymes that carry out the reversible reduction of protons into hydrogen gas, are of great interest, as they are often suggested for potential incorporation into a fuel cell and/or clean generation of hydrogen. By using proteins such as hydrogenases, which utilize cheap, abundant metals such as nickel or iron, we overcome the economic barrier of using rare earth metal catalysts such as platinum. However, these native hydrogenases are not well suited for general-purpose application due to their limited stability with respect to temperature and pH, rapid inactivation when exposed to oxygen, complexity of their biosynthesis, and limited potential for scalability and industrial application. Thus, we seek to accomplish chemistry similar to that performed by the [NiFe] hydrogenases by designing new functionality into a currently existing, robust metalloprotein.
Using Nature as inspiration, we seek to harness the advantages of
bioinorganic platforms while overcoming the limitations of large, fragile protein systems. One such platform is the small, electron-transfer protein rubredoxin (Rd). With its promiscuous metal-binding site, wide pH and thermal stability, and high recombinant expression yields, Rd is an optimum candidate for use in catalyst design. We will present results demonstrating that when the native, iron-bound protein is substituted with a nickel center, Rd can catalyze the reduction of protons electrocatalytically. Various techniques have been used to probe this activity and characterize catalytic intermediates, including protein film electrochemistry, electron paramagnetic resonance spectroscopy, and spectrophotometric redox titrations. Analogous to native hydrogenases, nickel-substituted Rd has also been shown to coordinate small organometallic moieties such as CO and CN-. By manipulating nickel- substituted Rd, we hope to create a robust metalloenzyme and obtain insight into the key parameters required for catalytic hydrogen production and oxidation.

References:
Slater, J. W.; Shafaat, H. S. Nickel-substituted rubredoxin is an effective electrocatalyst for hydrogen production 2015 (In review)

Keywords: Hydrogen, Metalloenzyme, Electrochemistry

107. Design and Characterization of Force-Sensitive DNA Origami Components

Yi Luo (Biophysics Graduate Program), Michael W. Hoduba (Department of Mechanical & Aerospace Engineering, The Ohio State University), Michael G. Poirier (Department of Physics, The Ohio State University), Carlos E. Castro (Department of Mechanical & Aerospace Engineering, The Ohio State University)

Abstract:
Scaffolded DNA origami is powerful design and fabrication tool for the creation of nanoscale objects via bottom up self-assembly. These objects have ~nm level geometric complexity and spatial accuracy, which is comparable to biological machinery. DNA origami has been used to create a wide range of objects such as drug delivery containers or platforms to guide molecular robots. Current applications of DNA origami exploit the large stiffness of bundles of dsDNA to create structures that maintain a well-defined and static geometry. However, DNA origami nanostructures with mechanically functional components, such as springs or actuators have remained largely unexplored. We aim to make DNA origami devices that are responsive to force magnitudes typically seen in biomolecular system (~picoNewtons). We have currently developed an approach to make force-sensitive DNA origami components and demonstrated this approach through the design of a two-state binary force sensor. This force sensor incorporates structures similar to DNA hairpins into DNA origami designs. The hairpin-like structures undergo a conformational change at a specific force threshold. We have characterized the conformational change dynamics of this force sensor using different experimental methods including single-molecule total internal reflection fluorescence microscopy, transmission electron microscopy and magnetic tweezers. We have shown that such dynamics can be tuned according to the design to meet the requirement of a wide range of applications. An analog force sensor is also in development using components that continuously deform under an applied force. Ultimately we aim to use these devices to measure forces of molecular interactions in cellular systems, for example cellular traction forces applied during cell migration.

Keywords: DNA Origami, Single-molecule, Self-assembly

108. Metabolic characterization of pennycress (Thlaspi arvense L.) for the production of valuable fatty acids

Enkhtuul Tsogtbaatar (Department of Molecular Genetics, The Ohio State University), Jean-Christophe Cocuron (Department of Molecular Genetics, Center for Applied Plant Sciences, The Ohio State University ), Marcos Corchado Sonera (Mechanical Engineering Department, University of Puerto Rico,), Ana Paula Alonso (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.