Talk Abstracts

Tuesday 09:15-09:30am: Assessing the role of ATF4-dependent signaling in limiting pancreatic cancer by tomatidine.

Debasmita Mukherjee (The James Comprehensive Cancer Center, Columbus, OH- 43210), Lena Bercz (The James Comprehensive Cancer Center, Columbus, OH- 43210), Molly Torok (The James Comprehensive Cancer Center, Columbus, OH- 43210), Timothy Pfau (The James Comprehensive Cancer Center, Columbus, OH- 43210), Thomas Mace (The James Comprehensive Cancer Center, Columbus, OH- 43210)

Abstract not available online - please check the booklet.

Tuesday 09:30-09:45am: ALS-associated KIF5A mutation causes delayed and decreased recovery following sciatic nerve crush.

Kelly A. Rich (Neuroscience Graduate Program), Meredith L. Jones (Neuroscience Undergraduate Program), Ashley Fox, Hallie Harris, Mehmet Yalvac (Department of Neurology, The Ohio State University Wexner Medical Center), Megan G. Pino (Neuroscience Graduate Program), W. David Arnold (Department of Neurology, The Ohio State University Wexner Medical Center), Stephen J. Kolb (Department of Neurology, The Ohio State University Wexner Medical Center)

Abstract:
Objective: KIF5A encodes kinesin protein KIF5A, which is involved in anterograde transport of cargos. C-terminal KIF5A mutations have been identified in patients with amyotrophic lateral sclerosis (ALS). The mechanism linking KIF5A mutations and ALS pathogenesis is unknown. Peripheral nerve injury (PNI) is an established paradigm to study mechanisms of axon repair, including axon transport. Degradation of the motor unit is a primary biological feature of ALS. We hypothesize that ALS-associated KIF5A mutations alter the ability of motor neurons to be maintained or repaired.

Methods: We created a novel mouse with an ALS-associated C-terminal Kif5a mutation (c.3005+1G>A; heterozygous [HET] Kif5a+/c.3005+1G>A; homozygous [HOM] Kif5a c.3005+1G>A/c.3005+1G>A). PNI was induced (sciatic nerve crush) in 14-week mice (wildtype [WT], HET and HOM). Mice were monitored via weekly electrophysiological assessments (compound muscle action potential [CMAP], motor unit number estimation [MUNE]), and unilateral hindlimb grip strength). At 16 weeks post PNI, we studied motor unit histopathology (motor neurons, sciatic nerve axons and neuromuscular junctions [NMJ]).

Results: We observed a loss of functional motor units in mutant mice compared to WT during recovery from PNI. On histopathology, we saw mutant-specific deficits in motor neurons, sciatic nerve axons and at the NMJ. Ipsilateral motor neurons showed decreased area in mutants (HET and HOM) as compared to WT. In sciatic nerve axons, we saw decreased axon fiber diameter in the ipsilateral nerves across all genotypes, however the decrease was most pronounced in mutant mice. At the NMJ, we saw decreased pre- and postsynaptic co-localization area as well as nerve terminal area in mutants.

Conclusions: We have defined a repair defect in motor axons with an ALS-associated KIF5A mutation. Our findings suggest Kif5a C-terminal mutations result in a loss of normal KIF5A function, impacting the motor unit. Mutant mice demonstrated observable pathological correlates throughout motor unit tissues. This work establishes a model system to develop disease- modifying therapies and understand basic principles of KIF5A biology.

Keywords: Amyotropic Lateral Sclerosis, Electrophysiology, Motor Unit

Tuesday 09:45-10:00am: Actives-Based Receptor Selection Strongly Increases Success Rate in Structure-Based Drug Design and Leads to Identification of 22 Unique Potent Cancer Inhibitors

Eric R Hantz (Biophysics Graduate Program), Steffen Lindert (Chemistry and Biochemistry Department, Ohio State University)

Abstract:
The drug discovery pipeline is plagued with inefficiency and false positive hits that has led to the pursuit of many experimentally inactive compounds. We have developed a methodology, based on the knowledge of known actives, to improve true positive prediction rates in structure-based drug design and have successfully applied the protocol to twenty unique target systems and identified a small ensemble of the top three performing conformers for each of the targets. Receptor performance was evaluated based on the area under the curve of the receiver operating characteristic curve for two independent sets of known actives. For a subset of five diverse cancer-related disease targets, we validated our approach through experimental testing of the top 50 compounds from a blind screening of the ChemBridge EXPRESS-Pick Collection using Glide SP. Our methods of receptor and compound selection resulted in the identification of 22 novel inhibitors in the μM-nM range, with the most potent inhibitor having an IC₅₀ value of 7.96 nM. Additionally for a subset of five independent target systems, we demonstrate the utility of Gaussian accelerated Molecular Dynamics (GaMD) to thoroughly explore a target system’s potential energy surface and generate highly predictive receptor conformations.

Keywords: Virtual Screening , GaMD, SBDD

Tuesday 10:00-10:15am: Identification of a tRNA-specific function for the tRNA methyltransferase Trm10 in Saccharomyces cerevisiae

Isobel Bowles (OSBP), Aiswarya Krishnamohan (OSU), Abi Hubacher (OSU), Jane Jackman (CBC OSBP)

Abstract:
tRNA methyltransferase 10 (Trm10) methylates N1 of guanosine at the 9th position of tRNA molecules using methyl donor S-adenosyl methionine (SAM). Upon deletion of trm10, Saccharomyces cerevisiae strains exhibit growth defects in the presence of antitumor drug 5-fluorouracil (5FU). We hypothesized that tRNA stability decreases with the lack of the m1G9 modification in trm10Δ strains and that certain tRNA species may be more reliant upon the methylated G9 nucleotide. We showed that when Trm10 substrate tRNATrp is overexpressed in trm10Δ strains, growth hypersensitivity to 5FU is rescued, while overexpression of 37 other tRNA species in S. cerevisiae does not rescue growth in the presence of the drug. We then demonstrated that levels of tRNATrp decrease in trm10Δ strains, and that these levels decrease further in the presence of 5FU, but another Trm10 substrate (tRNAGly) remains at a similar level in all conditions. These data indicate that the loss of m1G9 from tRNATrp is specifically responsible for the trm10Δ growth defect, even though Trm10 modifies 13 other tRNA substrates in S. cerevisiae. To identify the specific pathway associated with hypomodified tRNATrp quality control, pairwise deletion strains were created. These studies revealed that pairwise deletion of trm10 in combination with a known enzyme associated with multiple tRNA quality control pathways can rescue the 5FU-dependent growth defect. The effects of the m1G9 modification on tRNA structure and its interaction with Trm10 are also being determined with chemical footprinting methods, including SHAPE. Trm10 interactions with the tRNA at sites distant from the m1G9 modification were revealed that may play a role in the sensitivity of hypomodified tRNATrp. Together, these studies provide insight into the biological impact of loss of this highly conserved modification.

Keywords: tRNA modification, tRNA surveillance, SHAPE

Tuesday 10:35-10:55am: High-fat diet impairs long-term memory and synaptic plasticity in aged rodents via neuroinflammatory mechanisms

Ruth M. Barrientos (Department of Psychiatry & Behavioral Health; Institute of Behavioral Medicine Research, Ohio State University), Brigitte Gonzalez Olmo (Division of Anatomy, Ohio State University), Fangli Zhao, Candice Askwith (Department of Neuroscience, Ohio State University), Michael Butler ( Institute of Behavioral Medicine Research, Ohio State University), Stephanie Muscat (Biomedical Sciences Graduate Program, Ohio State University), James DeMarsh, Menaz Bettes ( Institute of Behavioral Medicine Research, Ohio State University)

Abstract not available online - please check the booklet.

Tuesday 10:55-11:15am: Biogenesis of tiny RNAs

GeunYoung Sim, (MCDB), Audrey Kehling, Mi Seul Park, Jackson Secor, Huaqun Zhang, Divyaa Bhagdikar (CBC), Ekram Abd El-Wahaband (CBC), Kotaro Nakanishi (CBC)

Abstract not available online - please check the booklet.

Tuesday 11:15-11:35am: New insights into the alternative translation initiation factor eIF2A

Daisy J. DaVita (Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Michael G. Kearse (Department of Biological Chemistry and Pharmacology, Center for RNA Biology)

Abstract:
Canonical eukaryotic mRNA translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) to deliver the initiator tRNA. However, eIF2A (not to be confused with the alpha subunit of eIF2) was the first initiator tRNA carrier discovered in eukaryotes, but further insights into its molecular mechanism dwindled once eIF2 was identified as the primary initiator tRNA carrier. The exact role of eIF2A in translation still remains a mystery despite it having nearly identical affinity for initiator tRNA and present at equal concentrations in cells as eIF2. Recent work has shown that eIF2A is required for cancer progression and proper long-term lipid metabolism in mice. Atypical of a translation factor, eIF2A primarily localizes to the nucleus, but can shuttle to the cytoplasm during cell stress. How and why eIF2A kept away from the translation machinery is unclear but it may be a mechanism for cells to only use eIF2A in specific conditions. To answer these questions, we first supplemented mammalian in vitro translation extracts with recombinant human eIF2A and found that excess recombinant eIF2A inhibits mRNA translation. To decipher which step of translation is inhibited, we used sucrose gradient ultracentrifugation along with various translation elongation inhibitors to capture and measure the levels of translation complexes at different stages in vitro. Using the non-hydrolyzable GTP analog GMPPNP to capture 48S initiation complexes (small subunit with eIFs at start codons), we identified that excess eIF2A reduces the abundance of small subunits at start codons. Excess eIF2A also inhibited translation directed by all four types of IRES elements, including those that do not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters the 40S subunit. Importantly, reactions supplemented with additional 40S subunits rescued translation. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering the 40S ribosomal subunit.

Keywords: ribosome, translational control, mRNA

Tuesday 11:35-11:55am: Nanocardiology: A Microscopy-Driven Approach to Cardiac Biology and Physiology

Rengasayee Veeraraghavan (Dept. of Biomedical Engineering, OSU)

Abstract:
Growing evidence indicates that cardiac biology and physiology at cellular through organ scales are governed by the action of proteins organized within nanodomains with specialized ultrastructural properties. Multiple phenomena have been identified, whose function and dysfunction cannot be predicted without accounting for the makeup and behavior of nanodomains. Thus, my laboratory’s investigative approach is grounded in high resolution structural and functional imaging, which is complemented by nanoelectrophysiology (Radwański), functional imaging (Györke) and computational modeling (Weinberg). I will present results from ongoing projects to illustrate how this multi-pronged approach is enabling us to take on biological and physiological questions ranging from basic science to translational.
1) The nuts and bolts of cardiac impulse propagation: Cardiomyocytes are electrically coupled via highly specialized and heterogeneous structures called intercalated disks. Using our novel indirect correlative light and electron microscopy (iCLEM) approach, we are compiling the first-ever quantitative picture of healthy and diseased intercalated disks including ultrastructure and molecular organization from nano- through micro-scales. Computational models built using these predict structure-function relationships underlying cardiac conduction.
2) Multiscale arrhythmia mechanisms in calmodulinopathy: Using a multiscale investigative approach, we have uncovered how the D96V mutation in calmodulin dysregulates NaV1.6 neuronal sodium channel inactivation and structural organization to promote pro-arrhythmic Na+/Ca2+ mishandling.
3) Distributed protein synthesis in cardiac myocytes: Using single molecule RNA visualization and spatial image analysis, we have discovered on-demand synthesis of membrane proteins at deployment sites from local mRNA pools provided by microtubule trafficking. We are now uncovering concurrent synthesis of multiple related proteins within microtranslatomes.

Keywords: Super-resolution Microscopy, Cardiovascular, Arrhythmia

Tuesday 03:00-03:15pm: Cryogenic Electron Microscopy Structure of an Aminopeptidase Reveals a Mechanism of Cooperative Assembly

Anthony Rish (OSBP), Tian-Min Fu (Department of Biological Chemistry and Pharmacology, Comprehensive Cancer Center, The Ohio State University)

Abstract:
Aminopeptidases are widely distributed in all kingdoms of life and play important roles in many physiological processes via cleaving amino acids from the amino terminus of proteins. The assembly of aminopeptidases is critical for their function and is quite diverse from species to species. Here, we present the cryogenic electron microscopy (cryo-EM) structure of B. subtilis aminopeptidase at 3.8 Å resolution. The aminopeptidase assembles into a dodecamer consisting of four trimers, in which each protomer is composed of seven alpha-helices and 15 beta-strands and the 12 protomers interact with each other via establishing two different types of interfaces: a dimeric interface and a trimeric interface. Unexpectedly, mutations of key residues in these two interfaces completely disrupt the oligomerization of the aminopeptidase, suggesting a cooperative assembly of B. subtilis aminopeptidase mediated by both the dimeric interface and the trimeric interface. Adopting these results, protein design techniques may be able to leverage the cooperative effects of interface interactions as a potential avenue for generating novel therapeutics.

Keywords: Cryo-EM, Aminopeptidase, Cooperative Assembly

Tuesday 03:00-03:15pm: Pnrc2-dependent mRNA decay and translational control mechanisms promote oscillatory gene expression during vertebrate segmentation

Monica C. Blatnik (Molecular, Cellular, and Developmental Biology), Thomas L. Gallagher (Department of Molecular Genetics), Sharon L. Amacher (Department of Molecular Genetics)

Abstract:
During early vertebrate embryogenesis, muscle and skeletal stem cells are grouped into reiterated segments, called somites, in a process called somitogenesis. Sequential somite formation is established by a genetic oscillator called the segmentation clock, comprised of a network of genes expressed periodically in the presomitic mesoderm. Precise control of segmentation clock oscillations is driven by robust temporal regulation of mRNA production, translation, and mRNA decay, and our work explores post-transcriptional mechanisms that regulate oscillatory expression. Proline-rich nuclear receptor coactivator 2 (pnrc2) regulates oscillatory mRNA decay in zebrafish embryos and loss of pnrc2 results in stabilization and accumulation of segmentation clock gene transcripts. However, pnrc2 mutant embryos exhibit normal segmentation clock protein expression and somite patterning is not disrupted. Polysome profiling was used to assess the translation state of segmentation clock gene transcripts her1, her7, dlc, and rhov, which revealed that all four transcripts were significantly increased in the ribosome-unbound (non-translating) state in pnrc2 mutants, compared to wildtype. Additionally, reporter analyses conducted to analyze the role of specific cis elements within the her1 3’UTR on mRNA stability and translation revealed that mutation of two regulatory elements, a Pumilio Response Element (PRE) and AU-rich Element (ARE), dramatically increased reporter transcript stability and polysome association, compared to the wildtype her1 3’UTR sequence. Pumilio and a subclass of ARE-binding proteins are known to promote mRNA decay and repress translation of their target transcripts. Our work investigating the role of RNA binding proteins, like Pumilio and ARE binding proteins, in regulating segmentation clock mRNA stability and translation will reveal how oscillatory gene expression is maintained to promote proper embryonic patterning.

Keywords: segmentation, oscillation, post-transcription

Tuesday 03:15-03:30pm: Analysis of tRNA methyltransferase homologs from zebrafish reveals similarities and differences to human Trm10 enzymes

Ben Jepson (MCDB)

Abstract:
The tRNA m1R9 methyltransferase (Trm10) family enzymes methylate the N-1 atom of purine residues at the ninth position of tRNAs. Higher eukaryotes encoding up to three homologs of Trm10. Human TRMT10A catalyzes m1G9 formation on multiple tRNAs, whereas TRMT10B forms m1A9 specifically on human tRNAAsp. Familial TRMT10A mutations are associated with human disease, further supporting the non-redundant function of the two homologs in vivo. We sought to develop a vertebrate model system to further probe the significance of these distinct activities using Danio rerio and have created mutant lines for the cytosolic homologs. Like human TRMT10A, expression of zebrafish Trmt10a rescues the trm10∆ growth phenotype in S. cerevisiae and methylates yeast tRNAs in vivo. Conversely, neither zebrafish Trmt10b nor human TRMT10B are capable of rescuing the phenotype or methylating yeast substrates in vivo. However, zebrafish Trmt10a and Trmt10b do not show the same pattern of in vitro substrate specificities as the human homologs. Since the modification status of zebrafish tRNAs had not been previously determined, we carried out a genome-wide study to map tRNA modification in zebrafish using mim-tRNAseq on wild-type embryos. We have now identified and validated multiple tRNA modifications in zebrafish, providing the first global map of tRNA modification in this organism, including target tRNAs for m1R9 modification. Analysis of R9 modification in the trmt10a and trmt10b mutant fish show that although each homolog acts on unique tRNA substrates similar to modification in humans, other substrates are shared between homologs, consistent with our in vitro experiments with purified enzymes. Overall, our data reveals that while the zebrafish enzymes share important similarities with their human counterparts there are distinct differences in some patterns of activity, deeper investigation of which could help uncover important insights into complex substrate specificities of Trm10 homologs.

Keywords: tRNA modification, Trm10, m1R9

Tuesday 03:15-03:30pm: Perineuronal net remodeling in the medial prefrontal cortex in a rodent model of postpartum depression

Courtney N. Dye (Neuroscience Graduate Program ), Dominic Franceschelli (Psychology Department ), Kathryn M. Lenz (Dept of Psychology, Dept of Neuroscience, Institute of Behavioral Medicine Research, The Ohio State University), Benedetta Leuner (Dept of Psychology, Dept of Neuroscience, The Ohio State University)

Abstract:
Postpartum depression (PPD) affects 20% of new mothers each year and is detrimental for both the mother and offspring’s wellbeing, but the mechanisms causing PPD are not fully understood. Utilizing a rodent model of gestational stress that induces depressive-like behaviors and impairs maternal care postpartum, I have been exploring the possibility that microglia, the brain’s innate immune cells, become perturbed in the PPD model to affect brain and behavior. Microglia activation can lead to the remodeling of perineuronal nets (PNNs), extracellular matrix proteins that regulate synaptic function. We hypothesized that remodeling of PNNs in the prefrontal cortex (PFC) a brain region that regulates mood and maternal behavior, plays an etiological role in the onset of PPD. Pregnant female rats either underwent stress from gestational day (GD)7-20 or were left undisturbed (controls), after which animals were sacrificed on either GD21 or postpartum day (PD)8 and brain tissue harvested. Immunofluorescent staining was conducted to visualize microglia and their phagolysosomes and PNNs. The number of PNNs and co-localization of PNNs with phagocytic microglia were quantified with ImageJ and Imaris software. Irrespective of stress condition, we found increased microglia engulfment of PNNs at GD21 compared to PD8, which corresponded with increased PNNs from late gestation to the postpartum period in control animals. We found a significant decrease in PFC PNN number in pregnant rats following exposure to stress compared to control rats at both GD21 and PD8. However, there was not an increase PNN engulfment by microglia across stress conditions at either timepoint. We show for the first time that pregnancy is associated with PNN remodeling in the PFC, and stress during pregnancy induced a loss of PNNs in the PFC. Given PNN loss was not mediated by microglia phagocytosis, future work will assess whether microglia regulate PNNs via inflammatory signaling. Overall, PNN remodeling in the PFC is a novel mechanism for plasticity in the maternal brain, and further remodeling following gestational stress may contribute to the underlying pathophysiology of PPD.

Keywords: Microglia , Perineuronal Nets , Maternal brain

Tuesday 03:30-03:45pm: IL1R1 knockout on glutamatergic neurons reverses RSD-induced alterations in fear conditioned behavior

Ethan Goodman (Department of Neuroscience), Samuel Swanson (Department of Neuroscience), Rebecca Biltz (Department of Neuroscience), Braeden Oliver (Department of Neuroscience), John Sheridan (Department of Neuroscience , Division of Biosciences-College of Dentistry), Jonathan Godbout (Department of Neuroscience)

Abstract not available online - please check the booklet.

Tuesday 03:30-03:45pm: The basis of multi-step tRNA recognition by a eukaryotic tRNA editing deaminase

Luciano G. Dolce (European Molecular Biology Laboratory, Grenoble), Aubree A. Zimmer, Juan D. Alfonzo (Ohio State Biochemistry Program), Laura Tengo (European Molecular Biology Laboratory, Grenoble), Felix Weis (European Molecular Biology Laboratory, Heidelberg), Henry Arthur, Mary Anne T. Rubio (Department of Microbiology, The Center for RNA Biology), Eva Kowalinksi (European Molecular Biology Laboratory, Grenoble)

Abstract:
Transfer RNAs (tRNAs) are central to protein synthesis by converting genomic information of protein-coding regions into a precise polypeptide chain, as dictated by codons in mRNA. tRNAs require extensive chemical modifications for structural stability, translation fidelity, as well as decoding functions. One such modification is the deamination of adenosine to inosine at the first position of the anticodon, which is essential for viability in bacteria and eukaryotes. Inosine formation at this wobble position in both bacteria and eukaryotes increases the decoding capabilities of a tRNA to recognize multiple different synonymous codons. The deaminase responsible for A-to-I activity in bacteria specifically deaminates a single tRNA while the eukaryotic counterpart has evolved to deaminate 7-8 tRNAs independent of any sequence specificity. How the eukaryotic enzyme recognizes multiple substrate tRNAs from non-substrate molecules has been a long-standing question in the field. In collaboration with the Kowalinski laboratory at the EMBL, we have described a model for tRNA recognition based on biochemical assays and the first structure of a eukaryotic deaminase bound to substrate tRNA. Our model describes a multi-step tRNA recognition mechanism using domains distal from the active site as well as an essential cation-π molecular gate guarding the catalytic site.

Keywords: tRNA editing, deaminase

Tuesday 03:45-04:00pm: The transcription factor Satb2 regulates pyramidal neuron integration into hippocampal circuits

Meretta A. Hanson (Neuroscience Graduate Program), Devipriyanka Nagarajan (Department of Neuroscience, Ohio State University College of Medicine), Alec H. Marshall (Department of Neuroscience, Ohio State University College of Medicine), Jason C. Wester (Department of Neuroscience, Ohio State University College of Medicine)

Abstract:
The CA1 region of the hippocampus contains pyramidal cells (PCs) that provide the output of hippocampal learning and memory computations to the rest of the brain. These CA1 PCs are heterogeneous and can be parsed based on laminar position into two subgroups: deep and superficial. Deep and superficial PCs are hypothesized to participate in different aspects of learning and memory due to their differential integration into hippocampal circuitry. For example, parvalbumin-expressing (PV+) basket cells preferentially inhibit deep PCs. This biased inhibition suppresses the output of deep PCs relative to superficial PCs in response to excitatory afferent input from neighboring hippocampal CA3. The mechanisms regulating the development of these different feedforward inhibitory circuits are unknown. Here, we test the hypothesis that the transcription factor Satb2 is critical for proper circuit integration of CA1 PCs. Satb2 is necessary for the differentiation of neocortical pyramidal cell types and is preferentially expressed in superficial PCs of the hippocampus. Using transgenic strategies in mice, we conditionally knocked out (cKO) Satb2 from pyramidal cells during early development. We then performed whole cell patch clamp recordings in acute hippocampal slices to examine the impact of Satb2 loss on PC integration into hippocampal circuits. We found that loss of Satb2 abolishes biased input from PV+ basket cells to deep PCs by preferentially increasing PV+ inhibition of superficial PCs. This increase in feedforward inhibition equalizes the responses of deep and superficial PCs to input from CA3. Thus, Satb2 is necessary for the proper differentiation of superficial PCs to maintain separate computations within CA1.

Keywords: Neural Circuits, Electrophysiology, Hippocampus

Tuesday 03:45-04:00pm: Identifying inhibitors of the FraB deglycase as therapeutics for Salmonella-mediated gastroenteritis

Jamison Law (OSBP), Kate Zaharova (Department of Biological Chemistry and Pharmacology, The Ohio State University), Anice Sabag-Daigle (Department of Microbiology, The Ohio State University), Austin Cool (Department of Chemistry and Biochemistry, The Ohio State University), Steffen Lindert (Department of Chemistry and Biochemistry, The Ohio State University), Charles Bell (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
Non-typhoidal Salmonella enterica (Salmonella) causes significant morbidity/mortality (193 million/85,000 worldwide in 2016) and economic costs ($11 billion in 2010, US alone) ^1,2. No vaccines are available, existing antibiotics are disfavored, and drug targets are scarce due to the pathogen’s versatility in exploiting redundancy/overlap in metabolic pathways ^3-9. However, fructose-asparagine (F-Asn) was recently discovered as a nutrient utilized by Salmonella via five proteins encoded in the fraRBDAE operon ¹⁰. Surprisingly, ΔfraB mutants could grow in glucose but not in glucose + F-Asn. This growth inhibition is due to the toxic buildup of 6-phosphofructose-aspartate, the substrate of FraB ¹¹. The absence of FraB in mammals and most members of the human microbiota makes FraB an attractive anti-Salmonella target ¹². Therefore, we conducted high-throughput cell-based and biochemical assays to identify FraB inhibitors. From screening 131,165 compounds in the biochemical assay, we found 72 and 28 compounds that inhibit FraB at 250 and 25 µM, respectively. While none of these hits inhibited wild-type Salmonella, 8 of them (excitingly!) inhibited the growth of ΔtolC Salmonella, a mutant whose efflux capability is dampened. These hits are now the focus of medicinal chemistry initiatives. To aid this objective, we also obtained a high-resolution structure (2 Å) of FraB. Unexpectedly, the proposed catalytic residues, E214 and H230, were separated by 24 Å with the C-terminal tail (residues 318-331) acting as a lid over the active site. Kinetic studies of the FraB Δ318-331 mutant revealed a 1.5-fold lower k_cat and a 150-fold higher K_m compared to the wild-type, yet the structure revealed that E214 and H230 were now only 11 Å apart. Molecular dynamics simulations revealed that the catalytic center (residues 224-240) was rendered more flexible upon removal of the C-terminal tail, supporting the notion of active site restructuring upon substrate binding. These findings bolster substrate/inhibitor co-crystallization prospects and help forge an exciting path towards Salmonella-specific therapeutics.

References:
1. Collaborators GDD. Lancet Infect Dis. 11 2018;18(11):1211-1228.
2. Scharff RL. J Food Prot. 2012;75(1):123-131.
3. Strugnell RA, et al. Curr Opin Microbiol. Feb 2014;17:99-105.
4. Martin LB. Curr Opin Infect Dis. Oct 2012;25(5):489-99.
5. Gopinath S, et al. Proc Natl Acad Sci USA. 2014;111(44):15780-15785.
6. Diard M, et al. Curr Biol. 2014;24(17):2000-2005.
7. Wiström J, et al. Ann Intern Med. 1992;117(3):202-208.
8. Becker D, et al. Nature. 2006;440(7082):303-307.
9. Steeb B, et al. PLoS Pathog. 2013;9(4):e1003301.
10. Ali MM, et al. PLoS Pathog. 2014;10(6):e1004209.
11. Sabag-Daigle A, et al. Sci Rep. 2016;6:28117.
12. Sabag-Daigle A, et al. Appl Environ Microbiol. 2018;84(5).

Keywords: Salmonella, crystallography, therapeutic

Tuesday 04:15-04:30pm: Simulations of Surface-Induced Dissociation of a Protein Dimer Essential for Hearing

Zachary D. Smith (Biophysics Graduate Program), Dalton Snyder (Department of Chemistry and Biochemistry, The Ohio State University), Yu-Fu Lin (Department of Chemistry and Biochemistry, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University ; Biophysics Graduate Program)

Abstract not available online - please check the booklet.

Tuesday 04:15-04:30pm: Title not available online - please see the booklet.

Ashley Francois (Molecular, Cellular and Developmental Biology MCDB ), Oscar Bermeo-Blanco, Richard Gumina (Department of Internal Medicine, Ohio State University Wexner Medical Center), Alessandro Canella, Lynn Marcho, Erin McGrail (Department of Physiology & Cell Biology, Ohio State University Wexner Medical Center), Thong Ba Nguyen, Yiqiang Zhang (Department of Anatomy & Biochemistry & Physiology, University of Hawaii at Manoa), Federica Accornero, Paul Janssen (Department of Physiology & Cell Biology, Ohio State University Wexner Medical Center), Bryan Whitson (Division of Cardiac Surgery, Ohio State University Wexner Medical Center)

Abstract not available online - please check the booklet.

Tuesday 04:30-04:45pm: To catch a killer: Delineating the mechanistic role of Siglec-6, a novel glycoprotein target, for the treatment of Chronic Lymphocytic Leukemia

Jessica Nunes (MCDB, The Ohio State University), Ann Ventura, Kevan Zapolnik (Internal Medicine, The Ohio State University), Liwen Zhang (Campus Chemical Instrument Center , The Ohio State University), John Byrd (Internal Medicine, University of Cincinnati), Christoph Rader (Department of Immunology and Microbiology, Scripps Research), Natarajan Muthusamy (Internal Medicine, The Ohio State University)

Abstract:
Sialic acid-binding immunoglobulin-like lectins (Siglec) are a group of glycoproteins that regulate innate and adaptive immune function via glycan recognition. Siglec-6 and its ligand, sialyl Tn (sTn), are both overexpressed in patients with chronic lymphocytic leukemia (CLL). Here, we show for the first time the functional relevance of Siglec-6 and sTn in cell adhesion and migration in CLL. Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ bone marrow stromal cells from CLL patients. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells (a CLL cell line) and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 knock-out (KO) MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a human Siglec-6 transgenic mouse model and crossed it with the well-known TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. At 9 months, Siglec-6 x TCL1 mice have a higher percentage of circulating leukemic cells when compared to TCL1 mice (n=4), suggesting that Siglec-6 may accelerate disease progression in CLL. Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating the in-vivo efficacy of Siglec-6/CD3 targeted bispecific antibody as well as identifying the leukemogenic role of Siglec-6 in CLL.

References:
Till, K. J., Lin, K., Zuzel, M. & Cawley, J. C. The chemokine receptor CCR7 and alpha4 integrin are important for migration of chronic lymphocytic leukemia cells into lymph nodes. Blood 99, 2977-2984, doi:10.1182/blood.v99.8.2977 (2002).
2 Burger, J. A., Burger, M. & Kipps, T. J. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 94, 3658-3667 (1999).
3 Hasan, M. K., Rassenti, L., Widhopf, G. F., Yu, J. & Kipps, T. J. Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells. Leukemia 33, 653-661, doi:10.1038/s41375-018-0306-7 (2019).
4 Choi, M. Y. et al. Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia. Cell Stem Cell 22, 951-959.e953, doi:10.1016/j.stem.2018.05.018 (2018).

Keywords: Siglec-6, Chronic Lymphocytic Leukemia, Bispecific antibody

Tuesday 04:30-04:45pm: The origins of asymmetries in microtubule organizing centers and their role in early C. elegans development

Shayne M Plourde (MCDB)

Abstract not available online - please check the booklet.

Tuesday 04:45-05:00pm: Outer nuclear membrane proteins SINE1 and SINE2 play differential roles in regulating stomatal dynamics in Arabidopsis thaliana

Morgan Moser (Molecular, Cellular, and Developmental Biology), Alecia Biel (Institute of Genomic Medicine, Nationwide Childrens Hospital), Norman R Groves (Department of Molecular Genetics, The Ohio State University), Iris Meier (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the booklet.

Tuesday 04:45-05:00pm: Novel HER2 Heterogeneity Metrics via High Dimensional Spatial Proteomics in Breast Cancer

David Tallman (MCDB, The Ohio State University), Harry Nunns (MultiOmyx Group, NeoGenomics, Fort Myers, FL), Anna Juncker-Jensen (MultiOmyx Group, NeoGenomics, Fort Myers, FL), Daniel G. Stover (Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center)

Abstract not available online - please check the booklet.

Tuesday 05:00-05:15pm: Troponin I phosphorylation is essential for cardiac reserve

Sarah L. Sturgill (Biophysics), Vikram Shettigar (Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University), Lorien G. Salyer (Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University), Elizabeth A. Brundage (Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University), Brandon J. Biesiadecki (Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University), Mark T. Ziolo (Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University)

Abstract:
In response to an increase in the body’s metabolic demand (e.g., exercise), the heart must increase its pumping performance to meet this demand. To achieve this increase, the heart relies on its cardiac reserve, which is the ability to increase its contractile and diastolic function, but the mechanism(s) responsible for cardiac reserve is poorly understood. The myofilaments are essential for contraction and relaxation with Troponin I (the inhibitory subunit of troponin, TnI) being a key regulatory protein. Studies have shown that TnI serine 23/24 (S23/S24) phosphorylation, the most abundant and important TnI phosphorylation, is a key mechanism for accelerating relaxation by decreasing Ca2+ sensitivity. The role of TnI in cardiac reserve is unknown. For this study, we thoroughly characterized the systolic and diastolic reserve in TnI S23/S24 phosphorylation-null transgenic mice (S23/S24 mutated to alanine, AA mice). Even with increased Ca2+ sensitivity, the AA mice exhibited normal function at resting heart rate and no difference in cardiac structure compared to wildtype. The Bowditch effect (i.e., an increase in contractile function with increasing heart rate) is essential to increase in vivo heart performance. To examine the role TnI S23/S24 phosphorylation in systolic and diastolic reserve, we assessed hemodynamics via left ventricular catheterization on the Bowditch effect by increasing heart rate from 240 to 420 beats per minute. Our data exhibited a clear loss of diastolic and systolic reserve in the AA mice. Since we observed a clear inability to increase systolic and diastolic function in AA mice, we performed speckle tracking echocardiography to more quantitatively investigate AA mice function. We observed that AA mice demonstrated normal systolic radial strain rate and impaired diastolic strain rate at resting heart rate, indicating a mild directional diastolic dysfunction. We conclude that TnI S23/S24 phosphorylation is essential for cardiac reserve by enhancing systolic and diastolic function. A blunted cardiac reserve leads to heart disease making TnI S23/S24 phosphorylation a potential therapeutic strategy.

Keywords: myofilament, cardiac reserve, troponin I

Tuesday 05:00-05:15pm: Title not available online - please see the booklet.

Ian F. Price (OSBP), Wen Tang (Biological Chemistry and Pharmacology)

Abstract not available online - please check the booklet.