Poster abstracts

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2. Ehrlichia sp. HF outer membrane proteins as vaccine candidates in a fatal human monocytic ehrlichiosis mouse model

Mariella A. Mestres-Villanueva (Molecular, Cellular, and Developmental Biology MCDB), Khem Budachetri (Department of Veterinary Biosciences), Tsian Zhang (Department of Veterinary Biosciences), Wenqing Zhang (Department of Veterinary Biosciences), Mingqun Lin (Department of Veterinary Biosciences)

Abstract:
Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes the emerging tick-borne disease, human monocytic ehrlichiosis (HME), a severe, influenza-like illness. The only treatment for HME is the broad-spectrum antibiotic Doxycycline and there is no vaccine available. Ehrlichia spp. lack conventional pathogen-associated molecular patterns, but surface proteins and virulence factors have been identified and their roles in transmission and disease have been studied. Entry-triggering protein of Ehrlichia (EtpE) is an invasin required for bacterial entry into human cells; the C-terminal (EtpE-C) binds DNase-X to induce receptor-mediated endocytosis. Outer membrane proteins OMP-1B and P28 are immunodominant major surface proteins and porins expressed by bacteria in ticks and mammals, respectively. Ehrlichia spp. possess Type IV secretion system (T4SS) and VirB2 proteins function as major pilus subunits, thus playing an important role in binding of the host cell membrane and secretion of T4SS effectors into the host cell cytoplasm. The objective of this study is to determine the vaccine potential of four Ehrlichia surface proteins in the mouse model of acute fatal HME, using Ehrlichia sp. HF (EHF). The genes encoding each protein were cloned into a protein expression vector, followed by expression and purification of the recombinant proteins. Five groups of immunocompetent mice were immunized with rEtpE-C, rOMP-1B, rP28, rVirB2-4 or sham-immunized with ISCOM alone and will be challenged with EHF in tick cells and infected ticks. At euthanasia, blood, spleen, and liver will be collected for analysis of bacterial load and cytokine expression levels via qPCR and RT-qPCR, respectively. The immune cell population in spleen will be analyzed by flow cytometry. This project will lead to a more thorough understanding of the pathogenicity and immunological response in fatal ehrlichiosis patients and on the potential of these antigens for vaccination strategies.

Keywords: Ehrlichia, Immunization, ISCOM

3. Deciphering cooperating genes in VGLL2-NCOA2 driven infantile rhabdomyosarcoma

Delia Calderon (Molecular, Cellular, and Developmental Biology Program, The Ohio State University), Sarah Watson, Olivier Delattre (Paris Sciences et Lettres PSL Research University, Paris, France), Collette LaVigne, Lin Xu, Whitney Murchinson, Dinesh Rakheja (UT Southwestern Medical Center, Dallas, United States ), Didier Surdez (Balgrist University Hospital, University of Zurich, Zurich, Switzerland ), Franck Tirode (Univ Lyon, Universit Claude Bernard Lyon 1, Cancer Research Center of Lyon, INSERM 1052, CNRS 5286, Centre LonBrard, Lyon, France ), James Amatruda (Cancer and Blood Disease Institute, Childrens Hospital Los Angeles, Los Angeles, United States )

Abstract not available online - please check the booklet.

4. Sarcomere generation is localized to intercalated disk and dependent on store operated calcium entry in a model of rapid onset (Takotsubo) hypertrophy

Casey M. Beard (Biophysics), Vladimir Bogdanov (Department of Physiology and Cell Biology), Benjamn Hernandez Orengo (Department of Physiology and Cell Biology), Galina Sakuta, Svetlana Tikunova (Department of Physiology and Cell Biology), Jonathan P. Davis (Department of Physiology and Cell Biology), Sandor Gyorke (Department of Physiology and Cell Biology)

Abstract:
To achieve hypertrophic growth, a cardiomyocyte must incorporate new sarcomeres within the cell. The location and manner of this growth during either physiologic or pathologic hypertrophy is uncertain. We have recently shown a relationship between hypertrophic processes and store operated calcium entry (SOCE) that occurs near the intercalated disk (ID) of cardiomyocytes. We therefore hypothesize that incorporation of new sarcomeres during hypertrophy occurs at the ID and is impacted by SOCE. We developed a novel mouse model possessing the gain-of-function mutation H109R+/- in the SOCE protein STIM1 that has been linked to human stress-induced cardiomyopathy, also called Takotsubo cardiomyopathy (TCM). STIM1 H109R+/- mice implanted with isoproterenol (ISO) osmotic minipumps developed left ventricular (LV) ballooning and reduced LV ejection fraction reminiscent of TCM and showed premature deaths. Cardiomyocytes derived from STIM1 H109R+/- evidenced a significant increase in length 2-3 days after ISO pump implantation. Confocal Ca2+ imaging in STIM1 H109R+/- myocytes showed increased SOCE localized to IDs. To determine the location of new sarcomere generation during hypertrophy, we employed a myofibril-integrated biomarker through introduction of GFP-tagged troponin C (TnC-GFP) in the described STIM1 H109R+/- mouse model. Myocytes were isolated 70 hours later and confocally imaged to determine regional concentrations of TnC-GFP associated with new sarcomeric growth. Our data suggest intense fluorescent signals localized at myocyte ends, consistent with higher expression of TnC-GFP adjacent to IDs. These results suggest that genetic upregulation of STIM1 under adrenergic stress can cause rapid-onset LV dilation similar to TCM. The longitudinal growth of myocytes under these conditions involves SOCE-dependent sarcomere addition to myocytes at IDs.

Keywords: store operated calcium entry, Takotsubo cardiomyopathy, sarcomerogenesis

5. Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

Paul J. Russell (Cellular, Molecular, and Biochemical Sciences Program, The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Michael G. Kearse (Cellular, Molecular, and Biochemical Sciences Program, The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Jacob A. Slivka (Department of Biological Chemistry and Pharmacology, Department of Computer Science and Engineering), Elaina P. Boyle (Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Arthur H. M. Burghes (Department of Biological Chemistry and Pharmacology)

Abstract:
It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5′ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5′ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

Keywords: translational control, uORF, mRNA stability

6. Excess eukaryotic initiation factor 2A plays a regulatory role in mRNA translation

Daisy J. DiVita (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Michael G. Kearse (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology)

Abstract:
Canonical translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) as the initiator tRNA (Met-tRNAiMet) carrier and as a central node of regulation. However, eIF2 was not the first Met-tRNAiMet carrier identified in eukaryotes. eIF2A (a monomer that is non-homologous to eIF2) was the initial Met-tRNAiMet binding protein discovered, but its role in translation was overshadowed after eIF2 was identified. Recent reports have shown that eIF2A is required for cancer progression, is not part of the canonical set of initiation factors, displays uncharacteristic GTP-independence for tRNA binding (unlike other tRNA-binding translation factors), and is able to stimulate initiation at CUG codons using Leu-tRNACUG; however, the exact function of eIF2A remains unknown. To determine how eIF2A functions in translation initiation, we programed in vitro translation extracts with recombinant human eIF2A. Our preliminary data show a significant decrease in reporter activity when recombinant eIF2A is present, suggesting eIF2A is an inhibitor of translation. Here we will pursue testing how eIF2A is inhibiting translation and whether this inhibition requires specific translation factors during certain stages of initiation. Together, these mechanistic insights will shed light on how non-canonical initiation factors are used and regulate eukaryotic translation.

Keywords: translational regulation, ribosome, initiation factors

7. Title not available online - please see the booklet.

Shrijan Khanal (MCDB), Kara M. Braunreiter (Division of Hematology, Department of Internal Medicine, Wexner Medical Center, The Ohio State University), Lotus Neidemire-Colley (Division of Hematology, Department of Internal Medicine, Wexner Medical Center, The Ohio State University), Natalie E. Sell (Division of Hematology, Department of Internal Medicine, Wexner Medical Center, The Ohio State University), Yandi Gao, Sonu Kalyan, Marie Goulard (Division of Hematology, Department of Internal Medicine, Wexner Medical Center, The Ohio State University), Parvathi Ranganathan (Division of Hematology, Department of Internal Medicine, Wexner Medical Center, The Ohio State University)

Abstract not available online - please check the booklet.

8. RNA polymerase IV defines heritable regulatory patterns

Benjamin P. Oakes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, Ohio 43210), Natalie C. Deans (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; Centers for RNA Biology and Applied Plant Sciences, The Ohio State University, Columbus, Ohio 43210), Jay B. Hollick (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; Centers for RNA Biology and Applied Plant Sciences, The Ohio State University, Columbus, Ohio 43210)

Abstract:
Paramutation is a behavior in which one parental allele facilitates a meiotically heritable regulatory change at the other1. This behavior occurs at specific alleles of multiple maize loci - including purple plant1 (pl1) - encoding transcriptional activators of flavonoid biosynthesis2. The Pl1-Rhoades (Pl1-Rh) allele can exist in a highly expressed reference state (Pl-Rh) or a repressed paramutant state (denoted Pl′ )3. Pl′ states often revert to Pl-Rh in rmr6-1 mutants4 deficient for the RNA polymerase (RNAP) IV largest subunit5 indicating that RNAP IV maintains the heritable information specifying Pl1-Rh paramutation. Because RNAP IV both sources 24-nucleotide (24nt) RNAs5 and conditions the heritable regulatory status of Pl1-Rh4,6, we hypothesize that RNAP IV, and potentially small RNAs (sRNAs), define the heritable regulatory status of other genes. To test this idea, seedling RNA-seq and corresponding sRNA-seq profiles of heterozygous BC5 progeny from sibling rmr6-1 mutant and Rmr6-B73 / rmr6-1 fathers were compared to identify heritable RNAP IV-dependent effects. We found the absence of paternal RNAP IV significantly altered the RNA abundances of 140 genes. Additionally, normalized read abundances of 711 24nt sRNA clusters were significantly distinct. We discovered that a significant number of genes with increased messenger RNA (mRNA) levels overlap with decreased 24nt sRNA clusters within 100, 50, and 10 kilobases upstream or downstream. These overlaps point to other epialleles, like Pl1-Rh, whose regulatory reprogramming in the absence of RNAP IV persists through meiosis. 10% of these overlapping sRNA clusters correspond to published putative cis-regulatory elements (CREs)7,8,9 while 90% correspond to regions not previously identified as putative CREs. Our findings suggest that Pol IV conditions heritable regulatory patterns of multiple genes and that heritable changes of 24nt sRNAs in the absence of paternal Pol IV may highlight undiscovered regions of regulatory importance.

References:
1. Brink Genetics (1958) | 2. Hollick Nat Rev Gen (2017) | 3. Hollick et al. Genetics (1995) | 4. Hollick et al. Genetics (2005) | 5. Erhard et al. Science (2009) | 6. Erhard et al. Plant Cell (2013) | 7. Oka et al. Genome Biology (2017) | 8. Ricci et al. Nat. Plants (2019) | 9. Lozano et al. G3 (2021)

Keywords: Epigenetic Inheritance, RNA Polymerase IV, small RNAs

9. Does spinal cord injury (SCI) impair pressure ulcer (PU) healing? A pilot study

Christopher P. Vadala (Neuroscience Graduate Program), Stephen Vidman, Angela R. Filous (Department of Neurology, The Ohio State University, Wexner Medical Center, Columbus, OH), Jan M. Schwab (Department of Neurology, The Ohio State University, Wexner Medical Center, Columbus, OH)

Abstract:
Background: SCI disrupts the communication between the central nervous and immune system and results in systemic immune dysregulation, which may contribute to the body’s inability to resolve inflammatory events. Resolution of inflammation is an active process that initiates the removal of inflammatory cells (efferocytosis) from the injury site and is required for effective wound healing. A prevalent complication following SCI are PUs, affecting about one in three patients. These highly preventable skin wounds arise from ischemic pressure and shear forces, and some can persist long-term. This leads to re-hospitalization and can be origins for disease and consecutive septic conversions.
Hypothesis: SCI directly blunts PU healing through a dysregulated immune response, resulting in skewed wound healing.
Methods: The direct effects of SCI on PU healing and immune responses will be assessed using a clinically relevant SCI PU mouse model (contusion, 75 kDyne) we have established. This model uses three 24-hour ischemia/reperfusion (I/R) cycles (12h ischemia x 12h reperfusion) using magnets (1012G) applied to the dorsal skin. This creates two distinct skin lesions, with induction beginning three days after SCI. Wound healing and inflammatory responses are assessed using histology and FACS analysis of the PU up to 42 days post-SCI.
Results: Histology demonstrated that the inflammatory response at the PU site is skewed, and wound healing is delayed in animals with SCI compared to sham. We determined and compared differences in epidermis and dermis thickness.
Conclusions: Our data shows that the SCI interferes with wound healing, applying an experimental, controlled PU model. Future experiments will analyze a disruption to resolution in PU and investigate chronic PU development and potential therapeutics to stimulate resolution. The results of these studies can be translated to clinical settings and will advance our understanding of wound healing mechanisms under SCI.

References:
WHO. Spinal cord injury. Published November 19, 2013. Accessed April 5, 2022. https://www.who.int/news-room/fact-sheets/detail/spinal-cord-injury
Prüss H, Kopp MA, Brommer B, et al. Non-Resolving Aspects of Acute Inflammation after Spinal Cord Injury (SCI): Indices and Resolution Plateau: Non-Resolving Inflammation. Brain Pathology. 2011;21(6):652-660.
Bannenberg GL, Chiang N, Ariel A, et al. Molecular Circuits of Resolution: Formation and Actions of Resolvins and Protectins. The Journal of Immunology. 2005;174(7):4345-4355.
Kumar S, Yarmush ML, Dash BC, Hsia HC, Berthiaume F. Impact of Complete Spinal Cord Injury on Healing of Skin Ulcers in Mouse Models. Journal of Neurotrauma. 2018;35(6):815-824.
Peirce SM, Skalak TC, Rodeheaver GT. Ischemia-reperfusion injury in chronic pressure ulcer formation: A skin model in the rat. Wound Repair and Regeneration. 2008;8(1):68-76.

Keywords: spinal cord injury, wound healing, pressure ulcer

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

Lucas A Runyan (Department of Chemistry and Biochemistry), Elena Kudryashova (Department of Chemistry and Biochemistry), Dmitri Kudryashov (Department of Chemistry and Biochemistry)

Abstract not available online - please check the booklet.

11. Identifying key amino acids differentiating the human and mouse Y145Stop prion protein fibril core structures by solid-state NMR

Christian ONeil (Biophysics), Theint Theint (Department of Chemistry at the Ohio State University)

Abstract:
The conversion of certain α-helical monomers to β-sheet-rich fibrils is determined to be the cause of many neurodegenerative diseases in humans and other animals.  To understand how the structure of prion amyloids correspond to the spread and development of these diseases, we first need to study how the primary sequence affects the overall fibril structure.  By comparing variants of prion protein Y145Stop, we can learn how their differences directly impact the conformation of the fibrils.  Solid-state NMR provides high-resolution images that can reveal small structural changes between samples as well as possible heterogeneity within one sample.  This technique is used to compare the human and mouse variants of prion protein which have only four sequence mutations near the fibril core. By recording spectra for each mutation, we can determine the role these residues play in the structure of the fibrils. Ultimately, this study will help us identify critical regions along the fibril core that determine its conformation.

References:
T. Theint, P.S. Nadaud, D. Aucoin, J.J. Helmus, S.P. Pondaven, K. Surewicz, W.K. Surewicz, C.P. Jaroniec, Species-dependent structural polymorphism of Y145Stop prion protein amyloid revealed by solid-state NMR spectroscopy, Nat. Commun. 2017, 8, 753.

T. Theint, Y. Xia, P.S. Nadaud, D. Mukhopadhyay, C.D. Schwieters, K. Surewicz, W.K. Surewicz, C.P. Jaroniec, Structural studies of amyloid fibrils by paramagnetic solid-state nuclear magnetic resonance spectroscopy, J. Am. Chem. Soc. 2018, 140, 13161–13166.

Keywords: prion, nmr, fibril

12. Progress towards the characterization of DNA origami by mass photometry and Orbitrap-based charge detection mass spectrometry

Nicholas Horvath (Department of Chemistry & Biochemistry), Wolfgang Pfeifer (Department of Mechanical & Aerospace Engineering), Enrique Ruiz (Department of Mechanical & Aerospace Engineering), Carlos Castro (Department of Mechanical & Aerospace Engineering), Vicki Wysocki (Department of Chemistry & Biochemistry)

Abstract not available online - please check the booklet.

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

Harper Smith (Biophysics Program), Songyu Dong (Ohio State Biochemistry Program), Marcos Sotomayor (Biophysics Program), Dmitri Kudryashov (Department of Chemistry and Biochemistry)

Abstract not available online - please check the booklet.

14. Towards Elucidating the Structure and Oligomeric State of the Membrane-Adjacent Domain of Cadherin-23, a Protein Essential for Hearing.

Qurat Ashraf (MCDB, The Ohio State University), Giovanna Grandinetti, Narui Yoshie, Elakkiya Tamilselvan, Travis Harrison-Rawn, Jasanvir Sandhu, Marissa Boyer, Marcos Sotomayor (The Ohio State University)

Abstract:
Mechanotransduction is the process by which hair cells of the inner ear transform mechanical stimuli into electrical signals. These electrical signals are transmitted to the central nervous system for processing in hearing and balance. Hair cells harbor actin-filled apical protrusions called stereocilia. Upon mechanical stimulation, fine “tip-link” protein filaments connecting the tip of a stereocilium to a neighboring taller stereocilium are stretched. As these filaments stretch, they pull and open ion channels to elicit an ionic current that initiates sensory perception. Tip-link filaments are made of homodimers of cadherin-23 (CDH23) and protocadherin-15 (PCDH15), two proteins involved in inherited deafness. CDH23 forms the upper two-thirds of the tip links and interacts with PCDH15 at the lower end to form a hetero-tetrameric complex. CDH23 and PCDH15, calcium-dependent cell-cell adhesion proteins, have multiple extracellular cadherin (EC) “repeats”, each about 100 amino acids long with a characteristic Greek-key fold. In addition to these EC repeats, both CDH23 and PCDH15 have membrane-adjacent domains, MAD28 and MAD12, respectively, which are predicted to be similar in structure. Intriguingly, biochemical assays and crystal structures of PCDH15 reveal that MAD12 induces parallel dimerization and that it has a ferredoxin-like fold, unlike the Greek key fold for EC repeats. Yet the structure and biochemical properties of MAD28 and of its adjacent CDH23 EC repeats are unknown. To determine whether CDH23 MAD28 has a ferredoxin-like fold that facilitates parallel dimerization, we tested several protocols to express and purify various CDH23 fragments that include EC25 to MAD28. We are now able to express and purify milligram amounts of well-refolded protein being used for biophysical characterizations using small-angle X-ray scattering and Cryogenic electron microscopy. To further our understanding of the role of MAD28 in mediating dimerization of CDH23, we introduced several mutations aimed to disrupt a possible MAD28 dimerization interface suggested by AlphaFold 2. Results from these experiments will advance our understanding of the role played by CDH23 in inner ear mechanotransduction.

References:
Jaiganesh, A. et al., (2018). Zooming in on cadherin-23: Structural diversity and potential mechanisms of inherited deafness. Structure, 25:1210-1225.
De-la-Torre, P. et al., (2018). A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15. Biophysics Journal, 115: 2368-2385.
Choudhary, D. et al., (2020). Structural Determinants of Protocadherin-15 Mechanics and Function in Hearing and Balance Perception. PNAS.

Keywords: Mechanotransduction, Tip link, Biophysical characterization

15. Visualization of the tip-link proteins involved in mechanotransduction of inner ear

Elakkiya Tamilselvan (Biophysics Graduate Program), Yoshie Narui (Center for Electron Microscopy and Analysis CEMAS, The Ohio State University), Giovanna Grandinetti (Center for Electron Microscopy and Analysis CEMAS, The Ohio State University), Daisy Alvarado (Biophysics Graduate Program), Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

16. Allosteric Coupling in an ATP-Driven Motor Protein

Rodrigo Muzquiz (OSBP), Kelly Karch (Department of Chemistry and Biochemistry, OSU), Vycki Wysocki (Department of Chemistry and Biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU ), Kristie Baker (Department of Chemistry and Biochemistry, OSU), Philip Lacey (Department of Chemistry and Biochemistry, OSU)

Abstract:
Hexameric helicases are motor proteins that play important physiological roles in cellular replication, chromosome packaging, and transcription termination3. The Escherichia coli (E.coli) Rho factor is an essential motor protein involved in regulating protein expression1. This ring-shaped protein binds nascent mRNA in its central pore via RNA binding loops. This facilitates movement along the ssRNA substrate to contact stalled RNA polymerase thereby terminating transcription. Functionally conserved residues in these loops make RNA contacts whose sidechain orientation correspond to different ATP-bound states1,3. The process of ATP and RNA binding leads to a conformational change from an open ring (lock-washer) to a closed ring. ATP hydrolysis occurs sequentially around the ring; however, it is not known how these protomers communicate to coordinate translocation. Therefore, understanding how the state of an ATP-bound site alters the conformation of an RNA-binding loop is crucial to characterizing AAA+ ATPase translocation. We have implemented native mass spectrometry (nMS) in tandem with surface induced dissociation (SID) to probe the allosteric and structural properties of Rho. nMS allows us to quantify the populations of each ATP-bound state of Rho rather than simply the solution ensemble. ATP titration experiments of apo-Rho have shown that there is negative cooperativity in binding. These experiments have also shown that Rho exists in variable oligomeric states (dimer, trimer, etc.) during the electrospray process that are not normally observed in solution under these same conditions. From these titration experiments we can fit the data to a statistical thermodynamic model to quantify coupling free energies. These coupling free energies would describe how the conformation of the RNA-binding loops is perturbed by nearest-neighbor interactions of ATP-bound protomers. In our next steps, we plan to optimize spraying conditions and nucleotide analogs to reduce the variability in oligomeric conformations observed so that the data fitting can yield more accurate parameters. We also seek to understand how the presence of RNA shifts the populations of ATP-bound states of Rho.

References:
1. Mitra P.; Ghosh G.; Hafeezunnisa M.; Sen R. Rho protein: roles and mechanisms. Annu Rev Microbiol. 2017, 8 (71) 687. DOI: 10.1146/annurev-micro-030117-020432
2. Yu Y.; Liu H.; Yu Z.; Witkowska H.E.; Cheng Y. Stoichiometry of nucleotide binding to proteasome AAA+ ATPase hexamer established by native mass spectrometry. Mol Cell Proteomics. 2020, 19 (12): 1997. DOI: 10.1074/mcp.RA120.002067
3. Patel, S. S., & Picha, K. M. (2000). Structure and Function of Hexameric Helicases. Annual Review of Biochemistry, 69(1), 651–697. https://doi.org/10.1146/annurev.biochem.69.1.651

Keywords: Allostery, ATPase, Mass-Spectrometry

17. Three-dimensional ultrastructural adaptation of central axons regulated by their mechanical microenvironment

Di Ma (OSBP), Binbin Deng (Center for Electron Microscopy and Analysis), Chao Sun (MCDB), David W McComb (Center for Electron Microscopy and Analysis), Chen Gu (Department of Biological Chemistry and Pharmacology)

Abstract:
Axon shafts mediate unidirectional conduction of action potentials and long-distance transport of proteins and organelles from neuronal soma to its synaptic terminals. How these long slender structures are regulated mechanically remains poorly understood. Combining confocal microscopy and cryo-electron tomography (Cryo-ET) with in vivo and in vitro systems, we report that nonuniform mechanical interactions with the microenvironment can lead to more than 10-fold diameter enlargement in an axon of the central nervous system (CNS). In the normal brain of adult Thy1-YFP transgenic mice, individual axons in the cortex displayed significantly higher diameter variation than those in the corpus callosum. When being cultured on lacey carbon film of electron microscopy (EM) grids, CNS axons formed varicosities preferentially in holes, with enriched mitochondria, multivesicular bodies (MVBs), and small vesicles, similar to axonal varicosities induced by mild fluid puffing. Microtubules (MTs) remained constant in all of our Cryo-ET varicosities and were asymmetrically separated at axon branch points often with de novo formation. When axons were fasciculated mimicking in vivo axonal bundles in white matter, varicosity levels reduced. Taken together, our results have revealed several novel features of three-dimensional ultrastructures of central axons in response to the nonuniform microenvironment.

References:
Browne, K.D., Chen, X.H., Meaney, D.F., and Smith, D.H. Mild traumatic brain injury and diffuse axonal injury in swine. J Neurotrauma 28, 1747-1755.
Gu, C. (2021). Rapid and Reversible Development of Axonal Varicosities: A New Form of Neural Plasticity. Front Mol Neurosci 14, 610857.
Hoffmann, P.C., Giandomenico, S.L., Ganeva, I., Wozny, M.R., Sutcliffe, M., Lancaster, M.A., and Kukulski, W. (2021). Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids. Elife 10.
Foster, H.E., Ventura Santos, C., and Carter, A.P. (2022). A cryo-ET survey of microtubules and intracellular compartments in mammalian axons. J Cell Biol 221.
etc.

Keywords: Axonal varicosity, Cryo-electron tomography (Cryo-ET), Axon

18. Defining the roles of biophysical membrane tension in breast cancer cell migration

Emily T. Chan (Biophysics), Travis H. Jones (Mechanical and Aerospace Engineering), Yasaman Madraki (Physics), Jonathan W. Song (Mechanical and Aerospace Engineering), Comert Kural (Physics)

Abstract not available online - please check the booklet.

19. Regulation of satellite cell pool maintenance by FGFR1 and FGFR4

Chung-Ju Yeh (Molecular, Cellular, and Developmental Biology Program)

Abstract not available online - please check the booklet.

20. Determining the role of the LINC complex in stomatal closure

Katelyn Amstutz (Molecular, Cellular and Developmental Biology), Norman Groves (Department of Molecular Genetics, The Ohio State University), Iris Meier (Department of Molecular Genetics, The Ohio State University; Center for RNA Biology, The Ohio State University)

Abstract:
As climate change worsens, droughts across the globe have increased in frequency and severity. Understanding how plants regulate and resist drought stress will allow us to better create crops resilient to the changing climate. Plants balance water loss with gas exchange through the opening and closing of pores called stomata, formed from two guard cells surrounding the central pore. In response to drought, plants produce the hormone Abscisic Acid (ABA), which causes a signaling cascade of Ca2+ and reactive oxygen species, resulting in a loss of turgor pressure and a filamentous actin (F-actin) rearrangement. Recently, loss of the Arabidopsis Klarsicht/ANC-1/Syne Homology (KASH) proteins AtSINE1 or AtSINE2 was shown to result in a defect in ABA-induced stomatal closure. KASH proteins form the outer nuclear membrane (ONM) component of Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, where they interact with inner nuclear membrane (INM) SUN proteins. KASH proteins have a cytoplasmic region which interacts with the cytoskeleton, while SUN proteins can interact with the nuclear lamina. However, whether these interactions are important for SINE1 or SINE2 function during stomatal closure is unknown. In addition, the role of SUN proteins and the nuclear lamina in stomatal closure has not been determined.
To examine which connections of SINE1 and SINE2 are required for stomatal closure, domain deletions of the SINE1 and SINE2 domains implicated in these interactions have been tagged with GFP and placed under their native promoters into sine1-1 and sine2-1 mutants, respectively. Localizations of these domain deletions have been obtained, and these lines will be examined for rescue of ABA-induced stomatal closure. If the LINC complex is required for the role of SINEs in stomatal closure, SUN and lamina mutants will be examined for defects in ABA-induced stomatal closure.

Keywords: LINC complex, Plant Biology

21. Role of the sarcin-ricin loop (SRL) of 23S rRNA in assembly of the 50S ribosomal subunit

Sepideh Fakhretaha Aval (Ohio state Biochemistry program, Department of Microbiology, Center for RNA biology, The Ohio State University), Kurt Fredrick (Ohio state Biochemistry program, Department of Microbiology, Center for RNA biology, The Ohio State University)

Abstract:
The sarcin-ricin loop (SRL) is one of the most conserved segments of the large subunit ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G and EF-Tu and IF2, interact with the SRL, and this interaction is essential for GTP hydrolysis. Cleavage and modification of the SRL by cytotoxins α-sarcin and ricin disrupt GTPase-ribosome interaction, leading to reduced protein synthesis and cell death. However, the full role of the of SRL remains unclear. A previous study showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. These ΔSRL ribosomes were purified from cells and found to be completely inactive in protein synthesis. Surprisingly, further analysis showed that ΔSRL ribosomes are not fully assembled. In particular, block 4 of the 50S subunit, which includes the components of the peptidyl transferase center, fails to fold. This defect could be due to loss of interactions between SRL and either (i) rRNA and/or ribosomal proteins or (ii) other (non-ribosomal) factors in the cell, such as trGTPases. Here, we describe in vitro 50S subunit reconstitution experiments and analysis of resulting particles. Our initial findings suggest that the defect is specific to ribosome biogenesis in the cell, consistent with the latter hypothesis.

References:
Moazed D, Robertson JM, Noller HF. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988;334(6180):362-364. doi:10.1038/334362a0
García-Ortega L, Alvarez-García E, Gavilanes JG, Martínez-del-Pozo A, Joseph S. Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding. Nucleic Acids Res. 2010;38(12):4108-4119. doi:10.1093/nar/gkq151
Lancaster L, Lambert NJ, Maklan EJ, Horan LH, Noller HF. The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit. RNA. 2008;14(10):1999-2012. doi:10.1261/rna.1202108
Davis JH, Williamson JR. Structure and dynamics of bacterial ribosome biogenesis. Philos Trans R Soc Lond B Biol Sci. 2017;372(1716):20160181. doi:10.1098/rstb.2016.0181

Keywords: Ribosome biogenesis, Sarcin-ricin loop, 23S rRNA

22. Determining the Relationship Between P-bodies and Tubulin Proteostasis

Wenfang Liu (MCDB program), Zachary Hurst (The Ohio State University), Shi Qian (MCDB program)

Abstract:
Processing-bodies (P-bodies) are subcellular ribonucleoprotein (RNP) granules that contain translationally-repressed mRNAs as well as proteins involved in mRNA decay. P-bodies are conserved from yeast to mammals and have been suggested to be sites of mRNA degradation and/or storage. These RNP granules are dynamic structures that form in response to a variety of stress conditions. We are interested in developing a better understanding of the regulation of P-body assembly and defining the physiological roles of these RNP granules. Towards these ends, we have identified a role for the heterohexameric chaperone complex, known as prefoldin, in P-body assembly. Specifically, we have found that the loss of prefoldin activity results in elevated P-body formation in both wild-type cells and a mutant that is normally defective for P-body assembly. Moreover, our data indicate that these effects are due to the defective folding of tubulin monomers and the ultimate disruption of the microtubule networks in the cell. As a result, we have proposed that there is a relationship between P-body assembly and tubulin proteostasis in eukaryotic cells.
Our current efforts are focused on characterizing these novel P-body-like foci and the underlying reasons that they are induced by microtubule disruption. Interestingly, these granules appear to differ from traditional P-bodies in several significant ways, including by granule morphology and overall protein composition. For example, we have found that several core P-body constituents are absent from these granules whereas tubulin monomers are present specifically in these structures. These differences as well as potential models to explain the physiological significance of these novel structures will be discussed herein.

References:
1. Thomas, M. G., Loschi, M., Desbats, M. A. & Boccaccio, G. L. RNA granules: The good, the bad and the ugly. Cell. Signal. 23, 324–334 (2011).
2. Luo, Y., Na, Z. & Slavoff, S. A. P-Bodies: Composition, Properties, and Functions. Biochemistry 57, 2424–2431 (2018).
3. Vainberg, I. E. et al. Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell 93, 863–873 (1998).
4. Gestaut, D. et al. The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis. Cell 177, 751-765.e15 (2019).
5. Sweet, T. J., Boyer, B., Hu, W., Baker, K. E. & Coller, J. Microtubule disruption stimulates P-body formation. RNA 13, 493–502 (2007).
6. Aizer A, Brody Y, Ler LW, Sonenberg N, Singer RH, Shav-Tal Y. The dynamics of mammalian P body transport, assembly, and disassembly in vivo. Chemtracts. 2008.

Keywords: RNP granules, P-bodies, tubulin proteostasis

23. PYM controls Exon Junction Complex occupancy at non-canonical positions

Manu Sanjeev (Molecular Cellular and Developmental Biology, OSU), Lauren Woodward (Department of Molecular Genetics, OSU), Robert Patton (Department of Physics, OSU), Ralf Bundschuh (Department of Physics,OSU), Guramrit Singh (Department of Molecular Genetics, OSU)

Abstract:
RNA processes such as transcription, splicing, export, translation, and decay are mediated by proteins that bind the RNA. The Exon Junction Complex (EJC) is a major RNA binding protein complex deposited ~24 nucleotides upstream of mRNA exon-exon junctions during the splicing reaction. The EJC remains stably bound to mRNA after splicing, modulates mRNA fate at multiple post-transcriptional steps, and is essential for viability in vertebrates [1]. A ribosome-associated factor called PYM, which binds the Y14/MAGOH heterodimer of the EJC core, is reported to aid in EJC disassembly during translation [2]. However, such a role for PYM is contradicted by the viability of Drosophila PYM null mutants [3] and sub-stoichiometric levels of PYM to both EJC and ribosomes. Here, we investigated the role of PYM in human cells by using a MAGOH mutant that assembles into EJC but is impaired in PYM interaction. Using a high-throughput footprint sequencing assay (RIPiT-Seq) in human embryonic kidney (HEK293) cells, we find that EJCs lacking PYM interaction show no defect in translation dependent disassembly but have increased footprints away from the -24 position (non-canonical EJC binding sites). Surprisingly, PYM interaction deficient EJCs are also enriched on unspliced transcripts from many single-exon genes that do not normally contain the EJC. PYM also has a dose dependent effect on the Nonsense Mediated mRNA decay (NMD) pathway. Our findings suggest a model for PYM function where PYM prevents spontaneous, splicing-independent deposition of EJC at non-canonical sites across the transcriptome. We are currently testing this model using biochemical and transcriptomic analyses. Our work reveals a potential mechanism for EJC binding at non-canonical sites and has important implications for viral pathogenesis as PYM is targeted by Kaposi Sarcoma-associated Herpes Virus to enhance viral RNA translation and by flaviviruses to promote viral replication via NMD inhibition [4].

References:
[1] L. A. Woodward, J. W. Mabin, P. Gangras, and G. Singh, “The exon junction complex: a lifelong guardian of mRNA fate: EJC: assembly, structure, and function,” WIREs RNA, vol. 8, no. 3, p. e1411, May 2017, doi: 10.1002/wrna.1411.
[2] N. H. Gehring, S. Lamprinaki, A. E. Kulozik, and M. W. Hentze, “Disassembly of Exon Junction Complexes by PYM,” Cell, vol. 137, no. 3, pp. 536–548, May 2009, doi: 10.1016/j.cell.2009.02.042.
[3] S. Ghosh, A. Obrdlik, V. Marchand, and A. Ephrussi, “The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila,” PLoS Genet, vol. 10, no. 6, p. e1004455, Jun. 2014, doi: 10.1371/journal.pgen.1004455.
[4] M. Li et al., “Identification of antiviral roles for the exon–junction complex and nonsense-mediated decay in flaviviral infection,” Nat Microbiol, vol. 4, no. 6, pp. 985–995, Jun. 2019, doi: 10.1038/s41564-019-0375-z.

Keywords: Exon Junction Complex, RNA decay

24. Title not available online - please see the booklet.

Andrea Keller (MCDB, Department of Biological Chemistry and Pharmacology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH), Amanda Cao (Harvard Medical School, Boston, MA), Maria Festing (Department of Biological Chemistry and Pharmacology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH), Vincenzo Coppola (Comprehensive Cancer Center, Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH), David M. Sabatini (Department of Biology, Massachusetts Institute of Technology MIT, Cambridge, MA), Maria M. Mihaylova (Department of Biological Chemistry and Pharmacology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH)

Abstract not available online - please check the booklet.

25. Determine the role of ZFP148 in regulating CD8+ T cell effector differentiation and exhaustion

Tong Xiao (MCDB program), Johanna Schafer (Pelotonia Institute for Immuno-Oncology, OSUCCC James), Anjun Ma (Department of Biomedical Informatics, OSU), No-Joon Song (Pelotonia Institute for Immuno-Oncology, OSUCCC James), Qin Ma (Department of Biomedical Informatics, OSU), Zihai Li (Pelotonia Institute for Immuno-Oncology, OSUCCC James)

Abstract:
CD8+ T cell effector differentiation and exhaustion in the tumor microenvironment is transcriptionally and epigenetically controlled by a number of transcription factors (TFs) such as T cell factor 1 (TCF1) and TOX. Dissecting how these intracellular factors program the above process is critical for the reinvigoration of exhausted cells. Through bioinformatic inference on a single-cell RNA-sequencing dataset acquired on CD8+ tumor-infiltrating lymphocytes (TILs), we identified several TFs to be active in a TCF1+ progenitor exhausted CD8+ T cell population, which potentially give rise to more exhausted progenies. Among these TFs, Zinc-Finger Protein 148 (ZFP148) showed one of the strongest positive correlations with Tcf7 (codes for TCF1). ZFP148 is a TF reported to regulate cell proliferation and survival in embryonic fibroblasts and other cell types but has not been studied in T cell biology. During preliminary investigations, I have found that ZFP148 expression could be induced by T cell receptor (TCR) engagement in mouse primary CD8+ T cells cultured in vitro and is positively associated with the activation status and effector function of the cells. Genetic deletion of ZFP148 lead to a more activated cell state and greater effector cytokine production. After evaluating its expression in CD8+ TILs, ZFP148 was found to be expressed at the highest level in terminal exhausted cell population, suggesting a role for ZFP148 in the development of CD8+ T cell exhaustion. Further, by combining CRISPR KO ZBP148 in CD8+ T cells and adoptively transferring into tumor-bearing mice, I observed that ZFP148-deleted CD8+ T cells were not able to control tumor growth as efficiently as control CD8+ T cells. To further dissect the precise mechanism by which ZFP148 regulates CD8+ T cell effector differentiation and exhaustion, I will (1) untangle the downstream molecular targets of ZFP148 in CD8+ T cells, (2) determine the role of ZFP148 in regulating the anti-tumor function of CD8+ T cell.

References:
1. Chen, Z., et al., TCF-1-Centered Transcriptional Network Drives an Effector versus Exhausted CD8 T Cell-Fate Decision. Immunity, 2019. 51(5): p. 840-855 e5.
2. Khan, O., et al., TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature, 2019. 571(7764): p. 211-218.
3. Im, S.J., et al., Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature, 2016. 537(7620): p. 417-421.
4. Merchant, J.L., et al., ZBP-89, a Kruppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter. Mol Cell Biol, 1996. 16(12): p. 6644-53.
5. Zou, Z.V., et al., Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway. Sci Rep, 2020. 10(1): p. 14156.

Keywords: Immuno-oncology, T cell exhaustion, Transcriptional regulation

26. Active mitochondrial fission during fungal biofilm formation

Hari Krishnan Balasubramanian (MCDB), Stephen A Osmani (Molecular genetics)

Abstract:
Fungal biofilms, formed by fungal cells that attach to a surface and grow, have unique characteristics including the presence of an extracellular matrix and resistance to anti-fungal drugs as well as the host immune system. As biofilms form they self-generate internal hypoxic microenvironments similar to that of tumors. Fungal biofilms therefore provide a unique opportunity to understand the mechanism of cell growth and survival in self-generated hypoxic/nutrient deprived conditions. Current understanding of the cell biology of biofilm forming cells is limited. In recent unpublished findings, we have seen mitochondria undergo fragmentation during fungal biofilm formation. A recent study demonstrated that upon deletion of DnmA and/or FisA, two core components of the mitochondrial fission machinery, mitochondria fail to undergo fragmentation after treatment with hydrogen peroxide in Aspergillus nidulans (Garrido-Bazán et al., 2020). To test if the mitochondrial fission machinery is required for the biofilm-formation induced fragmentation of mitochondria, we developed strains deleted for individual components of the mitochondrial fission machinery, namely DnmA, FisA and Mdv1. These strains show a loss of mitochondrial fragmentation during fungal biofilm formation, demonstrating a requirement of mitochondrial fission machinery for biofilm-formation induced mitochondrial fragmentation. Further, mitochondrial fragmentation is abrogated upon deletion of the hypoxic transcription factor SrbA, demonstrating that this process is under control of self-generated hypoxia during biofilm formation. Our future work will focus on understanding how loss of the ability to undergo mitochondrial fragmentation during biofilm formation affects other cellular biological modifications that occur during fungal biofilm formation, including dispersal of microtubules network and induction of autophagy (Shukla et al., 2017 and Lingo et al., 2021).

References:
Garrido-Bazán, V., Pardo, J. P., & Aguirre, J. (2020). DnmA and FISA mediate mitochondria and peroxisome fission, and regulate mitochondrial function, ROS production and development in aspergillus nidulans. Frontiers in Microbiology, 11.

Lingo, D. E., Shukla, N., Osmani, A. H., & Osmani, S. A. (2021). aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy. Molecular Biology of the Cell, 32(12), 1181–1192.

Shukla, N., Osmani, A. H., & Osmani, S. A. (2017). Microtubules are reversibly depolymerized in response to changing gaseous microenvironments within aspergillus nidulans biofilms. Molecular Biology of the Cell, 28(5), 634–644.

Keywords: Biofilm, SrbA, Hypoxia

27. Identifying motor proteins that function in male germ unit movement in Arabidopsis thaliana pollen tubes

Isabella Mendes (Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA, Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA ), Norman R Groves (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA, Center for Applied Plant Sciences, The Ohio State University, Columbus, OH, USA), Iris Meier (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA, Center for Applied Plant Sciences, The Ohio State University, Columbus, OH, USA, Center for RNA Biology, The Ohio State )

Abstract:
Fertilization is a key component of plant reproduction that is necessary for agriculture. While the basic process of fertilization has been understood for centuries, the mechanism underlying how plant sperm is trafficked to the egg remains poorly understood. To achieve fertilization, the growing pollen tube transports the sperm cell (SCs) to ovules. The SCs are physically connected via a cytoplasmic projection from the lead sperm cell to the vegetative nucleus (VN); this combined complex is referred to as the Male Germ Unit (MGU). Previous research has shown that Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes at the VN nuclear envelope facilitate MGU movement. The LINC complex spans the inner and outer nuclear membranes and connects the nucleoplasm to the cytoskeleton. Null mutants in genes for two plant LINC complex subunits, WIP and WIT, result in a VN movement defect, wherein the SCs lead the MGU through the pollen tube while the VN trails behind. This defect correlates with a defect in pollen tube burst and, in turn, a loss of seed set. We hypothesize that WIT and WIP act as adapter proteins between the VN envelope and unknown cytoskeletal motor proteins. In plants, there are two kinds of cytoskeletal motor proteins: Kinesins and Myosins. While Myosins have been well-studied in pollen-tube tip growth, the role of Kinesins in pollen tubes has not been established. To determine which motors are involved in VN movement, we have screened insertional mutants in 17 pollen-expressed Kinesins (PEK1-17) for fertility defects. Kinesin-14s represent a significant portion of the identified kinesins, with 8 of the 17 PEKs being Kinesin-14s. Two of these Kinesin-14s, PEK3 and PEK9, present mild fertility defects in insertional mutants. PEK3 and PEK9 are likely the result of a gene duplication event, leading to the possibility that a more severe fertility defect would be observed in a double mutant. Insertional mutants in a Kinesin-4, PEK14, displayed significant reductions in seed set, on the order of the reduction observed in wit mutants, indicating it may play a role in MGU movement. Future experiments will endeavor to determine the functional role of these motors in pollen and establish if these genes are associated with the VN envelope, physically interact with WIP and/or WIT, or are required for pollen tube burst.

References:
1. Zhou, X., and Meier, I. (2014). “Efficient Plant Male Fertility Depends on Vegetative Nuclear Movement Mediated by Two Families of Plant Outer Nuclear Membrane Proteins.” Proc. Natl. Acad. Sci. USA. 111:32, 11900-11905.
2. Zhou, X., et al. (2012). “Novel Plant SUN-KASH Bridges Are Involved in RanGAP Anchoring and Nuclear Shape Determination.” J Cell Biol. 196:2, 203-211.
3. Zhou, X., et al. (2015). “Plant Nuclear Shape Is Independently Determined by the SUN-WIP-WIT2-Myosin XI-i Complex and CRWN1.” Nucleus. 6:2, 144-153.
4. Cai G, Cresti M. (2009). “Organelle motility in the pollen tube: a tale of 20 years.” Journal of Experimental Botany. 60(2):495-508.
5. Heslop-Harrison J, et al. (1988) “Cytoskeletal elements, cell shaping and movement in the angiosperm pollen-tube.” Journal of Cell Science. 91:49-60.

Keywords: Male Germ Unit, Pollen Tube, Kinesin

28. RNA-binding proteins in cancer pathogenesis and as novel therapeutic targets

Joseph Mills (MCDB), Anna Tessari (Cancer Biology and Genetics), Eri Anastas (Cancer Biology and Genetics), Ilaria Cosentini (Cancer Biology and Genetics), Michael Kearse (DEPARTMENT OF BIOLOGICAL CHEMISTRY AND PHARMACOLOGY), Carlo Croce (Cancer Biology and Genetics)

Abstract not available online - please check the booklet.

29. Novel ACM Mouse Model Derived From a Human DSP Variant Displays a Cardiac Phenotype Upon Cardiac Stress

Tyler L. Stevens (MCDB), Heather Manring (Comprehensive Cancer Center, The Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH 43215, USA.), Trevor Dew, Michael J. Wallace, Aaron Argall, Sara Koenig, Mona El Refaey (2Department of Physiology and Cellular Biology, The Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH, 43215, USA), Xianyao Xu, Thomas J. Hund (Department of Biomedical Engineering, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43215, USA.), Maegen Ackermann (Department of Natural Sciences, Mansfield University of Pennsylvania, Mansfield, PA 17101, USA.), Peter J. Mohler (Physiology and Cellular Biology, The Ohio State University)

Abstract:
Arrhythmogenic Cardiomyopathy (ACM) is a disease that affects 1 in 2000 Americans yearly and segregates with sudden cardiac death (SCD). Approximately 60% of all ACM cases are associated with variants in desmosomal genes, however the molecular mechanisms associated with ACM remain unclear. Up to 20% of ACM-causing variants are within the desmosomal protein desmoplakin (DSP), a structural protein responsible for linking intermediate filaments to the intercalated disc (ID) to maintain structural integrity between cardiomyocytes. A mutational hotspot of ACM-linked variants exists within the N-terminus DSP, consisting of multiple spectrin repeats surrounding an SH3 domain. One studied DSP variant was identified in a large family that segregated strongly ACM, with multiple cases of SCD being identified. Tissue analysis from genotype-positive revealed fibro-fatty infiltration within the ventricular myocardium. Immunofluorescence identified disorganization of key ID proteins when compared to control tissue. Stem cells were isolated from the family to generate engineered heart tissue to identify changes in contractility and calcium signaling. Evaluation of the biomolecular properties of pathogenic hotspot-variants identified increased susceptibility to calpain-dependent degradation in select variants. Using molecular dynamics, we assessed essential intramolecular interactions and surface area exposure of a calpain target site within DSP to predict pathogenicity. To evaluate the effects of DSP variants on cardiac function and disease development, a mouse model containing a novel pathogenic DSP variant was established. Embryonic lethality was identified in mice carrying two copies of the novel variant. Mice expressing this novel variant heterozygously display reduced DSP protein levels from the ID. In addition, this novel mouse model displays an increased propensity for inducible ventricular arrhythmias upon adrenergic stimulation. Pressure overload resulted in reduction of cardiac functional output and chamber dilation. Future studies focus on establishing a mechanism of protection for calpain sensitive variants, along with further evaluation of the novel mouse model.

Keywords: Desmoplakin, Calpain, Arrhythmogenic Cardiomyopathy

30. Raman imaging to monitor lycopene uptake in cultured cells

Brian T. Scarpitti (OSBP), Chureeporn Chitchumroonchokchai (Department of Internal Medicine, The Ohio State University College of Medicine), Steven K. Clinton (Department of Internal Medicine, The Ohio State University College of Medicine), Zachary D. Schultz (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

31. Multimodal Magnetic Force Microscopy

Kevin J. Walsh (The Ohio State University), Joshua Sifford (The Ohio State University), Owen Shifflet (The Ohio State University), Tong Sheng (Rice University), Gang Bao (Rice University), Gunjan Agarwal (The Ohio State University)

Abstract:
Magnetic force microscopy (MFM) is a scanning probe technique that can detect nanoscale magnetic domains in a sample. MFM employs a magnetically coated probe to track the sample topography and detect long-range magnetic forces at user defined lift heights above the sample. Although MFM is widely used in solid-state devices, there are several challenges in application of MFM for biological samples. These include contamination of the MFM probe by sticky biological materials, topological cross-talk in MFM images and incompatibility in a fluid environment.
In this study we developed two methods to overcome the limitations of MFM and make it amenable to biological samples. In our first approach, we developed a novel indirect-MFM (ID-MFM) technique to detect fluorescently labeled iron-oxide nanoparticles. In ID-MFM an ultrathin silicon-nitride window is used to create a physical barrier between the sample and the probe. The window prevents direct contact between the sample and the probe thereby eliminating probe contamination and topological cross-talk. Finally, samples prepared for ID-MFM are amenable to multi-modal analysis by fluorescence or electron microscopy.
In our second approach we analyzed rodent spleen tissue sections via conventional MFM. We elucidate how scan rate and surface roughness can impact the topological cross-talk in MFM experiments which can make it difficult to decipher magnetic iron deposits. Thin sections with minimal surface roughness can minimize the topographical cross-talk. In addition, thin sections are compatible for multimodal microscopy analysis by both conventional (direct) and ID-MFM.
Taken together our work has advanced the use of MFM for biological samples. Future work along these directions could enable MFM analysis of physiological and pathological iron deposits in tissues sections in a label-free, multimodal and high throughput manner.

References:
Sifford, J., Walsh, K. J., Tong, S., Bao, G., & Agarwal, G. (2019). Indirect magnetic force microscopy. Nanoscale Advances, 1(6), 2348–2355. https://doi.org/10.1039/C9NA00193J

Keywords: magnetic force microscopy, iron, multimodal

32. Ultrafast dynamics of fatty acid photodecarboxylase in anionic semiquinone state

Ruiqi Wu (Biophysics Graduate Program, The Ohio State University), Chao Yang (Physics Department, The Ohio State University), Dongping Zhong (Biophysics Graduate Program, The Ohio State University)

Abstract not available online - please check the booklet.

33. Application of Multiscale Fragmentation Approaches to Enzymatic Barrier Heights

Paige E. Bowling (Biophysics), Dustin R. Broderick (Chemistry & Biochemistry, The Ohio State University)

Abstract:
This talk will explore the different aspects of fine-tuning multiscale fragmentation approaches for protein systems and then we will discuss its application for calculating enzymatic barrier heights. Recent work has shown that QM models with hundreds of atoms may be required to obtain convergence, which limits the levels of theory that can be applied. By using a multiscale fragmentation approach, we are able to push to the ab initio limit in systems with hundreds of atoms. Through sequential testing of distance-based thresholding, many-body expansion truncation, and different combinations of levels of theory for high-/low-level layers we have been able to establish a protocol for completing energetic calculations on protein systems. Here we have considered the methyl transfer by human catechol O-methyltransferase (COMT) to test our procedures capability to predict accurate enzymatic barrier heights.

Keywords: Quantum Chemistry, Computational Chemistry, Method Devlopment

34. Computational Studies of Molecular Force Sensors

Diana Lopez (Biophysics)

Abstract not available online - please check the booklet.

35. The PHF6 (VQIVYK) and related hexapeptides and the formation of Tau protein amyloid

Jaehoon Yang (Department of Chemistry and Biochemistry), Claire Hoffman (Molecular Biophysics Training Program), Mithila Agnihotri, Carol Husby, Jeff Kuret (Department of Biological Chemistry and Pharmacology), Sherwin J. Singer (Department of Chemistry and Biochemistry)

Abstract:
The VQIVYK fragment from the Tau protein, also known as PHF6, is essential for aggregation of Tau into neurofibrillary lesions associated with neurodegenerative diseases. VQIVYK itself forms amyloid fibrils composed of paired β-sheets. Therefore, the full Tau protein and VQIVYK fibrils have been intensively investigated. A central issue in these studies is polymorphism, the ability of a protein to fold into more than one structure. Using all-atom molecular simulations, we generate five stable polymorphs of VQIVYK fibrils, establish their relative free energy with umbrella sampling methods, and identify the side chain interactions that provide stability. The two most stable polymorphs, which have nearly equal free energy, are formed by interdigitation of the mostly hydrophobic VIY ‘‘face’’ sides of the β -sheets. Another stable polymorph is formed by interdigitation of the QVK ‘‘back’’ sides. When we turn to examine structures from cryo-electron microscopy experiments on Tau filaments taken from diseased patients or generated in vitro, we find that the pattern of side chain interactions found in the two most stable face-to-face as well as the back-to-back polymorphs are recapitulated in amyloid structures of the full protein. Thus, our studies suggest that the interactions stabilizing PHF6 fibrils explain the amyloidogenicity of the VQIVYK motif within the full Tau protein and provide justification for the use of VQIVYK fibrils as a test bed for the design of molecules that identify or inhibit amyloid structures.

Our studies of PHF6 revealed the importance of hydrogen-bond chains and π-stacking in stabilizing PHF6 fibrils. As a type of “gain of function” research, we constructed 11 hexapeptides which promised, and indeed we confirmed via simulations, to be even more stable than PHF6. These peptides have been evaluated in experiments, and the speed with which they form fibrils closely correlates with the calculated free energy of association.

Keywords: Molecular Dynamics , Amyloid Fibrils, Free Energy

36. Use of native mass spectrometry and mass photometry to elucidate structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein

Duc Phan (The Ohio State Biochemistry Program), Andrew S. Norris (Department of Chemistry and Biochemistry,Resource for Native Mass Spectrometry-Guided Structural Biology), Chen Du (Department of Chemistry and Biochemistry,Resource for Native Mass Spectrometry-Guided Structural Biology), Kye Stachowski (Department of Chemistry and Biochemistry), Vicki H. Wysocki (Department of Chemistry and Biochemistry,The Ohio State Biochemistry Program,Resource for Native Mass Spectrometry-Guided Structural Biology), Mark P. Foster, Venkat Gopalan (Department of Chemistry and Biochemistry,The Ohio State Biochemistry Program,Center for RNA Biology)

Abstract:
RNase P is a ribonucleoprotein (RNP) complex that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM structure of the Methanocaldococcus jannaschii (Mja) RNase P holoenzyme revealed unexpectedly a dimeric configuration, with each monomer of this archaeal variant containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, and L7Ae). Here, we used native mass spectrometry, mass photometry, and biochemical activity assays to (i) validate the oligomeric state of the Mja RNase P holoenzyme, (ii) report on a different stoichiometry for each holoenzyme monomer, where up to two copies of L7Ae are present, and (iii) identify the copies of L7Ae necessary for optimal cleavage activity. Moreover, by mutating all kink-turns (the binding sites for L7Ae) in the RPR, we made the surprising discovery that abolishing L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the safeguard provided by protein- protein interactions between L7Ae and other RPPs. Collectively, our results provide new insights into the architecture and evolution of RNase P, and highlight the value of using native MS and MP to augment the information obtained from low/medium-resolution cryo-EM structures

Keywords: RNase P, mass spectrometry, mass photometry

37. Elucidating the regulatory mechanism of FraR, a transcription factor in Salmonella enterica critical for pathogenesis

Blake E. Szkoda (The Ohio State Biochemistry Program, The Ohio State University), Anice Sabag-Daigle (Department of Microbial Infection and Immunity, The Ohio State University), Andrew Schweiters (Department of Microbiology, The Ohio State University), Brian Ahmer (Department of Microbial Infection and Immunity, The Ohio State University), Venkat Gopalan (Department of Chemistry & Biochemistry, The Ohio State University)

Abstract:
The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes approximately 94 million enteric infections and 50,000 diarrheal deaths annually worldwide.1 There are no vaccines or antibiotics that specifically combat this bacterium. During infection, Salmonella exploits fructose-asparagine (F-Asn) as a carbon and nitrogen source.2 F-Asn is a product of an Amadori rearrangement that occurs during cooking and dehydration of raw foods.3 F-Asn is metabolized by Salmonella using three enzymes and a transporter encoded by the fra operon. The roles of a periplasmic asparaginase, cytoplasmic kinase, and a cytoplasmic deglycase in F-Asn catabolism are now established.2,4 Importantly, the deglycase was identified as a promising drug target since its knock-out led to accumulation of its substrate and Salmonella self-poisoning.4,5 Our goal is to characterize regulation of the fra operon by FraR, the putative transcriptional factor in this locus. FraR is predicted to contain an N-terminal DNA-binding domain (DBD) and a C-terminal inducer-binding domain (IBD). We hypothesize that FraR binds to the fraB promoter in vivo, repressing the fra locus, and that binding of an inducer ligand to the FraR IBD triggers a conformational change to release the DNA from the DBD and permit transcription of the fra operon genes. Our recent biochemical and native mass spectrometry (nMS) studies with recombinant FraR provided insights into the inducer identity, the DNA and inducer binding affinity, and the binding stoichiometry.6 Building on these advances, we have identified key inducer binding residues in the IBD and the nucleotides in the DNA that are recognized by the DBD. To support our in vitro characterization, we are testing these mutants in vivo in vivo using a luciferase reporter system. Together, these data provide a model for how Amadori compound metabolism is regulated in a clinically significant bacterial pathogen and uncover thematic parallels in control of gene expression during utilization of unrelated nutrients.

References:
1) Majowicz et al. (2010) Clin. Infect. Dis. 50: 882-889
2) Ali et al. (2014) PLoS Pathog. 10: e1004209
3) Wu et al. (2017) J. Food. Ag. Chem. 66: 212-217
4) Sabag-Daigle et al. (2016) Sci. Reps. 6: 1-9
5) Sengupta et al. (2019) JMB 431: 4497-4513
6) Szkoda et al. (2022) JMB 434: 167480

Keywords: Salmonella, DNA, Amadori products

38. Substrate Recognition by Two 3′ to 5′ RNA Polymerases in Dictyostelium Discoideum

Grace Johnecheck (OSBP), Yicheng Long (OSBP), Jane Jackman (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

39. Determining the role of the 3’-5’ reverse polymerase in Myxococcus xanthus

Brandon Iwaniec (OSBP), Ashanti Matlock (OSBP), Jane Jackman (Department of Chemistry and Biochemistry )

Abstract:
tRNAHis guanylyltransferase-like proteins (TLPs) can catalyze nucleotide addition to RNA substrates in the opposite direction (3'-5') of all known canonical RNA polymerases. This biochemical activity is seen in vitro with bacterial TLPs, which can add nucleotides to the 5'-ends of tRNA substrates. However, no physiological relevant substrates have been identified to date in any bacterial species that has a TLP. Myxococcus xanthus, a bacterium, exhibits defects in starvation-induced fruiting body formation and sporulation when its MXTLP gene is deleted, suggesting that this enzyme plays a role in maturation or maintenance of one or more biologically relevant RNAs. We sought to test whether MxTLP catalyzes the addition of the essential G-1 nucleotide to the 5'-end of tRNAHis, which is an important function of some eukaryotic members of the tRNAHis guanylyltransferase (Thg1) family. This role for MxTLP was subsequently ruled in M. xanthus, based on primer extension assays of RNA isolated from MXTLP deletion vs. wild-type strains. This observation is consistent with the RNase P-dependent pathway for obtaining G-1 in bacteria, and the results of in vitro histidylation assays, in which we confirmed that M. xanthus histidyl tRNA synthetase prefers the form of G-1-containing tRNAHis that would most likely result from post-transcriptional RNase P cleavage, rather than from addition by MxTLP. Using a variety of in vitro biochemical assays, we sought to gain a more in-depth picture of the biological role of MxTLP in M. xanthus. We demonstrated that MxTLP can incorporate labeled nucleotides into specific RNA species isolated from vegetative and developing cells. Efforts to identify RNAs that are acted on by MxTLP are underway, and these results will be used to provide the first look at the potential biological function of a TLP in any bacterial system.

Keywords: Reverse polymerization , tRNA, Myxococcus xanthus

40. pre-piRNA trimming and 2′-O-methylation protect piRNAs from 3’ tailing and degradation in C. elegans

Benjamin Pastore (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University), Ian F. Price (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University)

Abstract:
The Piwi/piRNA pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2′-O-methylation at their 3’ termini. Here we report that 3’ termini of C. elegans piRNAs are subject to nontemplated nucleotide addition and piRNAs with 3’ addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2′-O-methyltransferase) accumulate piRNAs with 3’ nontemplated nucleotides. In henn-1 mutants piRNAs are shortened prior to 3’ addition whereas long isoforms of untrimmed piRNAs are preferentially modified in parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilencing of piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2′-O-methylation act collaboratively to protect piRNAs from tailing and degradation.

References:
Ozata, D.M., et al. (2019). PIWI-interacting RNAs: small RNAs with big functions. Nature reviews Genetics 20, 89-108.

Pastore, B., et al. (2021). pre-piRNA trimming and 2'-O-methylation protect piRNAs from 3' tailing and degradation in C. elegans. Cell Rep 36(9): 109640.

Gainetdinov, I., et al. (2021). Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability. Mol Cell 81(23): 4826-4842.

Keywords: piRNA , C elegans, PIWI Argonaute

41. E. coli Alanyl-tRNA Synthetase Maintains Proofreading Activity and Translational Accuracy under Oxidative Stress

Arundhati Kavoor (MCDB, OSU), Dr. Paul Kelly (MCDB, OSU)

Abstract:
Aminoacyl-tRNA synthetases (aaRS) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, therefore, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species (ROS) can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in E. coli AlaRS, unlike in other aaRSs studied to date, this does not affect proofreading activity against the non-cognate substrates serine and glycine, and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as ROS “sinks” to protect catalytically-critical sites from oxidative damage. Combined, our results indicate E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

Keywords: aminoacyl-tRNA synthease, tRNA, oxidative stress

42. Macrophage dynamics and disease progression in Duchenne Muscular Dystrophy

Natalie Aloi (MCDB), Geremy Lerma (Molecular Genetics), Joseph Beljan (MCDB), Jared Talbot (University of Maryland School of Biology and Ecology)

Abstract:
Duchenne Muscular Dystrophy (DMD) is a devastating and fatal disease affecting 1 in 5000 males. Although the standard of care for DMD is treatment with immunosuppressive drugs, the immunopathology of DMD is poorly understood. Understanding immune cell behaviors and immune-directed signaling over the course of the disease is critical to development of more targeted and effective therapies. Macrophages comprise a large portion of the immune infiltrate in DMD and can be polarized towards a myriad of activation states that are distributed across two broad categories: pro-inflammatory (M1), and anti-inflammatory (M2). M2-polarized macrophages secrete TGF-β, a pro-fibrotic cytokine implicated in dystrophic muscle damage. Paradoxically, it is also known that promoting M2 polarization improves muscle pathology in DMD models. It is unclear how M2 macrophages provide a protective effect in DMD despite producing TGF-β. We are characterizing the role of macrophages in vivo in a well-established zebrafish DMD disease model. Confocal analyses of dystrophic larvae reveal that macrophages are actively recruited to dystrophic lesions early and clear from the site of injury as lesions resolve, but that macrophage behaviors change as the disease progresses. Additionally, the number of TGF- β -responsive cells, indicated by high levels of phosphorylated (activated) SMAD3 protein (pSMAD3) is elevated near dystrophic lesions early in disease progression. These data support the hypothesis that macrophages and macrophage-directed TGF-β signaling play an important role in DMD disease progression.

References:
1.) McDouall RM, Dunn MJ, Dubowitz V. Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J Neurol Sci. 1990;99(2-3):199‐217.
2.) Kharraz Y, Guerra J, Mann CJ, Serrano AL, Muñoz-Cánoves P. Macrophage plasticity and the role of inflammation in skeletal muscle repair. Mediators Inflamm. 2013;2013:491497.
3.) Nguyen-Chi, M., Laplace-Builhe, B., Travnikova, J., et al. Identification of polarized macrophage subsets in zebrafish. ELife. 2015;4:e07288.
4.) Ceco E, McNally EM. Modifying muscular dystrophy through transforming growth factor-β. FEBS J. 2013;280(17):4198‐4209.
5.) Berger J, Currie PD. Zebrafish models flex their muscles to shed light on muscular dystrophies. Dis Model Mech. 2012;5(6):726‐732.

Keywords: Duchenne Muscular Dystrophy, Macrophage, Zebrafish

43. Role of reactive oxygen species in maternal hyperglycemia-induced congenital heart defects: a potential therapeutic target

Talita Choudhury (Center for Cardiovascular Research, Nationwide Childrens Hospital, Columbus, OH ), Sara Adamczak (Center for Cardiovascular Research, Nationwide Childrens Hospital, Columbus, OH ), Emily Morris Cameron (Center for Cardiovascular Research, Nationwide Childrens Hospital, Columbus, OH ), Madhumita Basu (Center for Cardiovascular Research, Nationwide Childrens Hospital, Columbus, OH ), Vidu Garg (Center for Cardiovascular Research, Nationwide Childrens Hospital, Columbus, OH )

Abstract:
Congenital heart defects (CHD) affect 1% of all live births in the US annually. The etiology of CHD is multifactorial, that is, both pathogenic genomic variation and environmental risk factors act as CHD contributors. Among the environmental teratogens, maternal pre-gestational diabetes mellitus (matDM) is associated with up to ~5-fold increase in the risk of having an infant with CHD. Generation of excess reactive oxygen species (ROS) due to maternal hyperglycemia is routinely observed in embryonic hearts exposed to matDM but the teratogenic effect of ROS on cardiac developmental pathways is not fully understood. The Notch and Nitric oxide (NO) signaling pathways, which are highly expressed in the endocardium, are critical for normal heart development. Previously, we identified a reduction in NO bioavailability and Notch signaling in mouse embryonic hearts exposed to matDM and reported a gene-environment interaction between Notch1+/- and matDM in the development of CHD. Here, we investigated the effect of modulating intracellular levels of oxidative stress in endocardial and endocardial-derived cell lineages to determine the role of ROS in matDM-associated CHD. Endocardial-derived cells exposed to oxidative stress elicit similar responses as hyperglycemia in reduction of NO bioavailability and Notch signaling. To study the role of oxidative stress in vivo, we generated WT and Notch1+/- embryos overexpressing the antioxidant gene, SOD1, to determine whether mitigating oxidative stress can rescue matDM associated CHD. We have confirmed a reduction of oxidative stress in SOD1-tg embryonic hearts and preliminary data shows lower incidence of CHD in SOD1-tg compared to WT embryos exposed to matDM. The role of oxidative stress in matDM-associated CHD requires further investigation, as mitigation of endocardial oxidative stress may be a promising approach to lower the incidence of CHD in high-risk populations.

References:
Basu M, Zhu JY, LaHaye S, Majumdar U, Jiao K, Han Z, Garg V. Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease. JCI insight. 2017 Oct 19;2(20).

Keywords: congenital heart defects, maternal diabetes, oxidative stress

44. Translation Initiation Factor Eif4e1c Drives Cardiac Growth During Development and Regeneration in Zebrafish

Anupama Rao (Molecular, Cellular, and Developmental Biology), Ishrat Jahan (Department of Biological Chemistry and Pharmacology, The Ohio State University), Baken Lyu (Department of Biological Chemistry and Pharmacology, The Ohio State University), Kenneth Poss (Department of Cell Biology, Duke University ), Kedryn Baskin (Department of Cell Biology and Physiology, The Ohio State University), Joseph Aaron Goldman (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
The mammalian heart lacks the capacity to regenerate damaged tissues making heart disease the leading cause of death in the world. However, zebrafish can regenerate large portions of cardiac tissues lost following injury. Many studies have investigated transcriptional changes occurring during regeneration to identify molecules and pathways driving this process. Here, we show that translation initiation factor, eif4e1c is required for efficient zebrafish heart development and regeneration. The eIF4E family of translation initiation factors bind 5’ methylated caps and act as rate limiting factors to determine mRNA translation. Deletion of eif4e1c in zebrafish using CRISPR resulted in lower survival rates to adulthood and overall growth deficits. Adult hearts had fewer cardiomyocytes (CMs) in eif4e1c mutants and CM proliferation was impaired during regeneration. Previous studies have hypothesized lower mitochondrial activity to be pro-proliferative. Surprisingly, we found that mitochondria in uninjured eif4e1c mutant hearts have lower activity even though CM proliferation is impaired in development. Lower mitochondrial activity was also observed in regenerating wildtype hearts in line with previous literature. Taken together, mitochondrial respiration appears to have both, pro-growth and inhibitory effects on heart muscle depending on context and the developmental stage. In conclusion, our study supports a model of cardiac regeneration in which translation is a critical component of regulating gene expression to drive CM proliferation.

References:
1. Poss, K.D., Wilson, L.G., and Keating, M.T. (2002). Heart Regeneration in Zebrafish. Science 298, 2188–2190.
2. Meyer, K.D., Patil, D.P., Zhou, J., Zinoviev, A., Skabkin, M.A., Elemento, O., Pestova, T.V., Qian, S.-B., and Jaffrey, S.R. (2015). 5’ UTR m(6)A Promotes Cap-Independent Translation. Cell 163, 999 1010.
3. Leppek, K., Das, R., and Barna, M. (2017). Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat Rev Mol Cell Bio 19, 158 174.
4. Hinnebusch, A.G., Ivanov, I.P., and Sonenberg, N. (2016). Translational control by 5’-untranslated regions of eukaryotic mRNAs. Science 352, 1413 1416.
5. Liu, Y., Beyer, A., and Aebersold, R. (2016). On the Dependency of Cellular Protein Levels on mRNA Abundance. Cell 165, 535–550.

Keywords: Cardiac regeneration, Translation, Zebrafish

45. Exploring the roles of protein cofactors in the conformational dynamics and regulation of RNase P

Milo Datta (MCDB), Ila Marathe (Post doctoral candidate, OSU)

Abstract:
tRNAs are adapter molecules playing crucial role in cellular translation. For these molecules to be functional, multiple enzymes process the premature tRNAs into the final mature form. RNase P is one such processor, catalyzing Mg2+ dependent 5′ maturation of precursor tRNAs (pre-tRNAs). The ribonucleoprotein (RNP) form of the enzyme utilizes a catalytic RNA aided by proteins. Across the spectrum of life, different numbers of RNase P Proteins (RPPs) associate with the RNase P RNA (RPR) to form the functional holoenzyme. Archaea possess up to 5 RPPs which share high homology with their eukaryotic counterparts who possess up to 10 RPPs. This makes the archaeal system a good model to biochemically explore the roles of the proteins in the RNP assembly and function. In archaea, the proteins are: RPP21, RPP29, POP5, RPP30 and L7Ae. RPP21•RPP29 and POP5•RPP30 form binary subcomplexes that bind the RPR, helping in substrate binding and catalysis respectively. Recent structural studies showed that RPR of Pyrococcus furiosus (Pfu), an archaeon, undergoes Mg2+ induced structural remodeling, through different paths, in the presence or absence of the RPPs. Effect of individual RPP components on the RPR structural reorganization is unknown. Furthermore, characterization of the RPPs in metal ion affinity and RPR binding is still required. The proposed study in the archaeal RNase P system seeks to unravel the contributions of RPPs in RPR conformational dynamics and regulation. Single molecule experiments will give insights into the role of RPPs in RPR binding and folding and cleavage assays with individual suites of RPP binary complexes will parse their roles in metal ion binding. The studies will help gain deeper insights into the archaeal RNase P enzyme and a better understanding of the system in eukaryotes.

References:
1.Phan HD, Lai LB, Zahurancik WJ, Gopalan V. The many faces of RNA-based RNase P, an RNA-world relic. Trends Biochem Sci. 2021;46:976-991.
2.Dreyfuss G, Philipson L, Mattaj IW. Ribonucleoprotein particles in cellular processes. J Cell Biol. 1988;106:1419-1425.
3.Ellis JC, Brown JW. The RNase P family. RNA Biol. 2009;6:362-369.
4.Esakova O, Krasilnikov AS. Of proteins and RNA: the RNase P/MRP family. RNA. 2010;16:1725-1747.
5.Lai LB, Vioque A, Kirsebom LA, Gopalan V. Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects. FEBS Lett. 2010;584:287-296.
6.Chen WY, Pulukkunat DK, Cho IM, Tsai HY, Gopalan V. Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex. Nucleic Acids Res. 2010;38:8316-8327.

Keywords: tRNA processing, RNase P, conformational dynamics

46. Molecular insights into different photorepair efficiencies of DNA Photolyases

Debanjana Chakraborty (OSBP), Chao Yang (Department of Physics), Lijuan Wang (Department of Physics)

Abstract not available online - please check the booklet.

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

Philip Kauffman (Ohio State Biochemistry Program)

Abstract not available online - please check the booklet.

48. Discovering SMAD3 Independent Pathways to Overcome TGF-b Mediated Immunosuppression in NK cells Against High-grade Gliomas

Ezgi Elmas (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH. ), Yasemin Sezgin (CRISPR/Gene editing core, Abigail Wexner Research Institute at Nationwide Childrens Hospital, Columbus, OH.), Meisam Naeimi Kararoudi (CRISPR/Gene editing core, Abigail Wexner Research Institute at Nationwide Childrens Hospital, Columbus, OH, Department of Pediatrics, The Ohio State University, Columbus, OH.), Dean A. Lee (Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Childrens Hospital, Columbus, OH, Department of Pediatrics, The Ohio State University, Columbus, OH. )

Abstract not available online - please check the booklet.

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

Hsiang-Yin Hsueh (MCDB), Zobeida Cruz-Monserrate (Division of Gastroenterology, Hepatology, and Nutrition, Division of Internal Medicine)

Abstract not available online - please check the booklet.

50. Pathogenesis of Two Newly Discovered Virulence Factors of Ehrlichia japonica, A Model Bacterium for Studying Fatal Human Monocytic Ehrlichiosis

Rory C. Chien (Department of Veterinary Biosciences, College of Veterinary Medicine ), Mingqun Lin (Department of Veterinary Biosciences, College of Veterinary Medicine ), Yasuko Rikihisa (Department of Veterinary Biosciences, College of Veterinary Medicine )

Abstract:
Human monocytic ehrlichiosis (HME) is an emerging, tick-borne febrile illness that potentially causes life-threatening infections. The causative pathogen, Ehrlichia chaffeensis, is a unique, obligate intracellular bacterium that primarily targets monocytes and macrophages. The in vivo pathogenesis of severe or fatal HME is not completely understood. Our laboratory developed a fatal HME mouse model by culture isolating Ehrlichia japonica, a bacterium closely related to E. chaffeensis, which causes severe diseases and death in immunocompetent mice due to cytokine storm and toxic shock-like syndrome. By using Himar1 transposon random mutagenesis system, we recently identified two novel virulence factors of E. japonica (EHF_0962 and EHF_RS04100) in the mutants H59 and H43B, respectively. Although these virulence factors are dispensable in vitro for infecting macrophage (DH82), endothelial (RF/6A), and tick (ISE6) cell lines, the H59 and H43B mutants cannot rapidly replicate and spread systemically to kill immunocompetent mice. My hypothesis is that the mutants lost the ability to overcome in vivo innate immune responses such as the reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by phagocytes. To investigate the virulence loss of the mutants, we will establish a time-coursed infection in the mouse model and design specific experiments for testing host innate immune responses. Furthermore, we will generate rescue plasmids to restore the virulence gene in the mutants for the complementation assays. Successful completion of these aims will provide critical knowledge of treating and preventing severe HME.

Keywords: Ehrlichia chaffeensis , Human monocytic ehrlichiosis , Virulence factors

51. Small RNAs Physiological Diffusion Dynamics in the Vasculature

Archie Bhullar (Biophysics ), Xin Li (Pharmacy, Ohio State University), Daniel Binzel (Pharmacy, Ohio State University), Peixuan Guo (Pharmacy, Biophysics, Ohio State University)

Abstract:
Using Oligonucleotides as biopolymer material in the medical application of nanostructures for drug delivery, as a targeting aptamer, or as a drug such as miRNA, siRNA, or mRNA, requires unique biophysical characterization in vivo for full utilization in the medical field. Among these unique biophysical elements, is the characterization of chemically modified small RNAs trans endothelial vasculature diffusional flux. The diffusional flux of small RNAs in several areas of the vasculature including the ectopic tumor vasculature and glomerular membrane in the kidneys are clinically relevant. Here I provide a preliminary rationale and methodology for the investigation of RNA trans-vasculature flux dependent on vasculature composition, pressure, and temperature. Furthermore, I propose the rationale for the design of a nanoparticle carrier with a thermodynamically unstable core and pseudoknot helix arms for flexible stoichiometry when coupled with the dynamic nature of RNA structure in vivo.

Keywords: RNA, Diffusion

52. Title not available online - please see the booklet.

Ruo-Wen Chen (Ohio State Biochemistry Program), Michael G. Poirier (Physics Program)

Abstract not available online - please check the booklet.

53. Role of Fibroblast Growth Factor Receptor-1 in mediating growth and survival in Mantle Cell Lymphoma

Anuvrat Sircar (Molecular, Cellular and Developmental Biology Graduate program), Satishkumar Singh (Division of Hematology, Department of Internal Medicine, The Ohio State University), Evangelia Chavdoula (The Ohio State University Comprehensive Cancer Center), Philip N. Tsichlis (The Ohio State University Comprehensive Cancer Center), Lalit Sehgal (Division of Hematology, Department of Internal Medicine, The Ohio State University)

Abstract:
Mantle Cell Lymphoma (MCL) is an aggressive form of non-Hodgkin’s lymphoma, with late-onset, high progression rates, and extensive bone marrow (BM) involvement. Despite various treatment strategies, survival has not improved in MCL. Targeted therapy using ibrutinib improves overall survival; however, relapse occurs, with 30-50% of patients presenting with de-novo or acquired resistance (AIR), illustrating a desperate need for alternative druggable targets and therapy options. Our unbiased integrated whole transcriptome analyses identified Fibroblast Growth Factor Receptor-1 (FGFR1) as a novel mutually upregulated candidate gene in ibrutinib-resistant patient B-cells, patient-derived cells cultured under stromal support, and patient-derived cells compared to healthy B-cells. Consistent with our analysis, conditioned media from the BM stroma confers cellular proliferation and tumor survival via the FGFR1 signaling pathway. Using a wide array of techniques such as ChIP-enrichment analysis, RNA Sequencing, ChEA, ArCHS4, GO & KEGG pathway analysis, overexpression/knockdown, and rescue studies, along with in-vivo FGFR1 inhibition experiments utilizing MCL AIR and PDX models in both immunodeficient and immunocompetent mice backgrounds, we show that FGFR1 expression modulates decreased sensitivity to ibrutinib via regulation of the PRC2 complex, particularly EZH2, in conjunction with KDM2B, a histone demethylase. Furthermore, we show that the drug sensitivity and tumorigenicity are modulated downstream through EZH2 controlled effect on CDKN family members. Our data demonstrate that FGFR1 can be therapeutically targeted in MCL to attack ibrutinib resistant tumors and a better understanding of the molecular signaling involved in this process, a stepping-stone towards developing effective targeted therapies to cure patients with relapsed MCL.

Keywords: Mantle Cell Lymphoma, Bone-marrow microenvironment, Drug resistance

54. A novel RNA based gene therapy approach to modulate gene expression for treatment of severe neurological disorders caused by mutations in FOXG1

Taylor Feldt (MCDB)

Abstract:
FOXG1 syndrome is a newly classified syndrome that causes developmental delays and structural abnormalities in the brain due to de-novo heterozygous mutations in the FOXG1 gene. FOXG1 syndrome was previously thought to be a congenital form of Rett’s syndrome and has since been reclassified as a new syndrome with its own unique phenotype. Unlike Rett’s syndrome, children affected by mutations in this gene never achieve developmental milestones, and instead have developmental delays and abnormalities that begin in utero. This is likely due to the crucial role the FOXG1 gene plays in development of the telencephalon. The FOXG1 gene is highly conserved and plays a non-redundant role in neural development and is reused at different times in different locations in the developing brain. As a newly classified syndrome, not much is known about the role the FOXG1 gene plays in disease pathology however, current research has discovered mutations in FOXG1 are typically unique to each patient. FOXG1 syndrome has proven to be an intellectually puzzling syndrome that could lead to innovative solutions and new treatments. The goal of this project is to better classify the FOXG1 gene itself, including promoter regions and the impact of mutations within different functional domains. The lab aims to achieve this through bioinformatics techniques as well as the development of an in-vitro model system for the syndrome using patient derived fibroblasts. This will allow us to determine the unique effects of mutations within certain defined functional domains of the FOXG1 gene. Further, the use of a FOXG1 in-vitro model will be crucial in the testing of established small drug molecules as potential new therapeutics to better fit the needs of patients.

References:
Akol I, Gather F, Vogel T. Paving Therapeutic Avenues for FOXG1 Syndrome: Untangling Genotypes and Phenotypes from a Molecular Perspective. International Journal of Molecular Sciences. 2022; 23(2):954. 
Dastidar, S. G., Landrieu, P. M., & D'Mello, S. R. (2011). FoxG1 promotes the survival of postmitotic neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(2), 402–413.
Mitter, D., Pringsheim, M., Kaulisch, M. et al. FOXG1 syndrome: genotype–phenotype association in 83 patients with FOXG1 variants. Genet Med 20, 98–108 (2018).
Murphy et al., 1994; Li et al., 1995; Bourguignon et al., 1998 – FOXG1 structure & function
Ni Y, Liu B, Wu X, Liu J, Ba R, Zhao C. FOXG1 Directly Suppresses Wnt5a During the Development of the Hippocampus. Neurosci Bull. 2021 Mar;37(3):298-310.

Keywords: FOXG1 , Gene Therapy , RNA Reprogramming

55. Virus reactivation in tumor boosts NK cell mediated cell cytotoxicity

Zhenyu.Wu (MCDB Program), Dawei.Zhou (Department of Pathology), Guillaume.Fiches (Department of Pathology), Taiwei.Li (MCDB Program)

Abstract not available online - please check the booklet.

56. Title not available online - please see the booklet.

Salma Abdelbaky (The Ohio State University ), Brian Giacopelli (The Ohio State University ), Kari G. Rabe (Mayo Clinic), Yue-Zhong Wu (The Ohio State University ), John C. Byrd (The Ohio State University ), Shanafelt, Sameer Parikh , Neil E. Kay, Esteban Braggio, Susan L. Slager (Mayo Clinic)

Abstract not available online - please check the booklet.

57. Mechanisms of Synergy between Oncolytic Herpes Simplex Virus and Trabectedin in Pediatric Bone Sarcomas

Emily Franz (Molecular, Cellular, and Developmental Biology), Matthew Cannon (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases), Pin-Yi Wang (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases), Amy Gross (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases), Maren Cam (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases), Mark Currier (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases)

Abstract not available online - please check the booklet.

58. Identify tumor intrinsic epigenetic regulators potentiating immune checkpoint blockade by MHC class I upregulation

Weiwei Liu (Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, The Ohio State University), Lei Zhou (Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, The Ohio State University), Tony Kwon (Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, The Ohio State University)

Abstract not available online - please check the booklet.

59. Oncolytic virotherapy can be augmented by inhibiting TGFbeta and anti-PD1 in immunogenic murine MPNST

Siddhi N. Paudel (Graduate Program in Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH, USA), Brian Hutzen (The Abigail Wexner Research Institute at Nationwide Childrens Hospital Center for Childhood Cancer and Blood Disorders, Columbus, OH, USA), Chun-Yu Chen (The Abigail Wexner Research Institute at Nationwide Childrens Hospital Center for Childhood Cancer and Blood Disorders, Columbus, OH, USA), Katherine E. Miller (The Abigail Wexner Research Institute at Nationwide Childrens Hospital Institute for Genomic Medicine, Columbus, OH, USA), Timothy P. Cripe (Division of Pediatric Hematology/Oncology/BMT, Department of Pediatrics, The Ohio State University Wexner College of Medicine, Columbus, OH, USA)

Abstract not available online - please check the booklet.

60. Role of Autotaxin in Central Nervous System Autoimmunity

Cora L. Petersen (Neuroscience Graduate Program ), Yue Liu (Department of Microbial Infection and Immunity, The Ohio State University), Shawn Murphy (Department of Microbial Infection and Immunity, The Ohio State University), Joshua Deffenbaugh (Department of Microbial Infection and Immunity, The Ohio State University), Amy Lovett-Racke (Department of Microbial Infection and Immunity, The Ohio State University)

Abstract:
Background:
Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS). A defining characteristic of MS is the ability of autoreactive T lymphocytes to cross the blood brain barrier (BBB) and mediate inflammation within the CNS. Previous study from our lab has found the gene ENPP2 to be highly upregulated in encephalitogenic T cells in the mouse model of MS. ENPP2 codes for the secreted protein autotaxin which promotes transendothelial migration of T cells from the blood stream into the lymphatic system. Our hypothesis is that inhibiting autotaxin signaling may prevent autoreactive T cells from crossing the BBB and causing neuroinflammation.

Aims:
Aim 1: Do MS patient CD4+ T cells show differential expression of autotaxin compared to healthy controls?
Aim 2: Does pharmacological inhibition of autotaxin ameliorate the mouse model of MS, experimental autoimmune encephalomyelitis (EAE)?

Method:
Peripheral blood mononuclear cells from MS patients and healthy controls were activated and analyzed for changes in autotaxin expression via flow cytometry. For EAE studies, mice were immunized to induce EAE and then treated with autotaxin inhibitor HA-130. Changes to disease severity were measured by tracking clinical score.

Results/Conclusions:
Our data show that MS patients’ CD4+ T cells differentially express autotaxin compared to healthy controls. In the context of EAE, we have found that treatment with autotaxin inhibitor HA-130 decreases EAE severity but does not affect the ability of CD4+ T cells to clear viral infection, suggesting autotaxin inhibition may be a viable therapeutic target.

Keywords: Neuroimmunology, Autoimmunity, Multiple Sclerosis

61. Chronic inflammation occurs in both the cochlea and the cochlear nucleus during age-related hearing loss

Benjamin J. Seicol (NGP), Shengyin Lin (Department of Otolaryngology, College of Medicine), Ruili Xie (Department of Otolaryngology, College of Medicine)

Abstract:
Age-related hearing loss (ARHL) is an increasingly common age-related pathology characterized by structural changes in the auditory pathway including the cochlea and cochlear nucleus (CN). Cochlear macrophages and CN microglia provide host defense and tissue surveillance. Both are long-lived cell populations that respond to environmental insults to promote and resolve inflammation. Failure to resolve inflammation leads to chronic inflammation that can enhance age-related pathology. While it is known that cellular and tissue damage following acoustic insults are worsened by acute inflammation, the impact of chronic inflammation during ARHL remains unclear. We hypothesized that accumulation and activation of cochlear macrophages and CN microglia occurs during ARHL in mice corresponding with loss of function. To test this, we investigated inflammation in the cochlea and CN of young, middle-aged, and aged CBA/CaJ mice. All mice were tested for auditory brainstem response (ABR) to assess hearing status. Cochlea and cochlear nucleus were collected and immunohistochemistry was used to label tissue with markers targeting ionized calcium binding adaptor molecule 1 (Iba1) and CD68—a marker of phagocytic activity. Confocal microscopy and quantitative image processing were used to measure the accumulation and activation of both cell populations across the lifespan. We found ABR thresholds increase during aging consistent with late-onset ARHL in CBA/CaJ mice. We also observed progressive increases in the area covered by Iba1-labeled macrophages in the osseous spiral lamina (OSL) that correlated with increased ABR threshold during aging. Notably, we found significant accumulation and activation of cochlear macrophages in middle-aged mice, which have relatively normal ABR threshold and prior to overt ARHL. CD68-labeled area increased during aging in both the OSL and CN indicating activation. C1q deposition increased during ARHL in the CN. Our study showed that chronic inflammation occurs in both the cochlea and the CN during aging, even in middle age prior to overt ARHL. These findings suggest that chronic inflammation may precede significant tissue damages of the auditory system and contribute to the development of ARHL.

Keywords: Macrophages, Microglia, Age-related Hearing Loss

63. Maternal allergic inflammation has sex-specific effects on oxytocin fiber density in brain regions associated with juvenile social play in rats

Michaela Breach (Neuroscience Graduate Program), Habib E. Akouri (Department of Psychology, The Ohio State University), Claire M. Dodson (Department of Psychology, The Ohio State University), Kathryn M. Lenz (Department of Psychology, Department of Neuroscience, Institute for Behavioral Medicine Research, The Ohio State University)

Abstract:
Background: Maternal immune activation (MIA) increases the offspring’s risk of neurodevelopmental disorders.1 Our rat model of acute allergic MIA produces offspring with symptoms akin to those found in neurodevelopmental disorders, including impaired juvenile social play behavior.2 Here, we further analyzed the behavioral profile found in this model and investigated whether MIA affected oxytocin (OT) and arginine vasopressin (AVP), two neuropeptides crucial for the regulation of social behavior. Methods: Virgin female rats assigned to the MIA group were sensitized to ovalbumin, bred, and allergically challenged on gestational day 15. Control females were administered saline on the same schedule. Male and female offspring were either tested for behavior (ultrasonic vocalizations, allogrooming, and social preference) or euthanized on postnatal day (P)28 for OT and AVP immunofluorescence analysis. OT and AVP neurons were counted in the paraventricular nucleus (PVN), while fiber staining was quantified in the lateral septum (LS), nucleus accumbens (NAc), and directly lateral to the PVN, in the lateral hypothalamic area (LHA). Results: Allergic MIA reduced neonatal USV frequency (F1,36 = 5.6, p = 0.02, eta2 = 0.13). In contrast to our published findings of impaired juvenile play, MIA did not impair sociability in adults (F1,34 = 0.009, ns). Interestingly, MIA reduced OT fiber density in the LS (F1,19 = 7.03, p = 0.02, eta2 = 0.24) and LHA (F1,17 = 5.22, p = 0.04, eta2 = 0.23) of juvenile males but not females. AVP fiber density was unaffected in the LS and LHA (F’s ≤ 2.13, ns). We are currently analyzing other brain regions relevant to sociality, including NAc and PVN. Conclusions: Allergic MIA may impair juvenile play behavior through sex-specific actions on oxytocinergic innervation of the social brain. Future work will investigate the immunological mechanisms underlying oxytocin alterations and social play deficits.

References:
1. Han, V.X., et al., Maternal acute and chronic inflammation in pregnancy is associated with common neurodevelopmental disorders: a systematic review. Transl Psychiatry, 2021. 11(1).

2. Breach, M. R., Dye, C. N., Joshi, A., Platko, S., Gilfarb, R. A., Krug, A. R., ... & Lenz, K. M. (2021). Maternal allergic inflammation in rats impacts the offspring perinatal neuroimmune milieu and the development of social play, locomotor behavior, and cognitive flexibility. Brain, Behavior, and Immunity, 95: 269-286.

Keywords: Maternal Inflammation, Oxytocin, Social Behavior

64. Sphingosine 1-Phosphate Receptor 1 Inhibition Rescues Muller Glia Reprogramming in the Microglia-Depleted Avian Retina

Liv Taylor (Neuroscience Graduate Program), Snehi Shah (Biomedical Science Undergraduate Program), Andy Fischer (Department of Neuroscience)

Abstract not available online - please check the booklet.

65. Title not available online - please see the booklet.

Athena Howell (Neuroscience)

Abstract not available online - please check the booklet.

66. Immediate induction of varicosities by transverse compression but not uniaxial stretch in axon mechanosensation

Chao Sun (MCDB, The Ohio State University), Lin Qi (Department of Biomedical Engineering, The Ohio State University), Yang Cheng (Department of Biomedical Engineering, The Ohio State University), Yi Zhao (Department of Biomedical Engineering, The Ohio State University), Chen Gu (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
Mild traumatic injury (mTBI) in mice damages axons and results in axonal varicosities, which manifest as bead-like swelling of the axonal shaft and can impede signal transduction. This study aimed to assess how the orientation of mechanical shock affects the quantity of axonal varicosities. Using Thy1-YFP transgenic mice, we visualized axonal varicosities in CHIMERA, a mouse model that mimics mild traumatic injury. Our results also show that axonal varicosities predate microglia activation to be the earliest known event for axonal injury. We found that axons perpendicular to the direction of the CHIMERA impact are more prone to varicosities than those parallel to the direction of impact, suggesting that transverse compression is more efficient than lateral compression when inducing axonal varicosities. Additionally, in cultured neurons, we found that axonal varicosities were more likely to be induced by transverse compression from a fluid puffing test than uniaxial strain from a nanowrinkle stretch assay. The finding provides insight for the better understanding of axonal mechanosensation. Potentially, clinicians can pay more attention to the impact direction in which a human patient receives a concussion and its effects to the tissue damage.

Keywords: Axonal varicosities, Traumatic brain injury, Mechanosensation

67. Amplified gliosis and interferon-associated inflammation in aged mice following diffuse traumatic brain injury

Lynde M. Wangler (Neuroscience Graduate Program), Chelsea E. Bray (College of Medicine, OSU), Jonathan M. Packer (Neuroscience Graduate Program), Zoe M. Tapp, Amara C. Davis (Neuroscience Graduate Program), Shane M. ONeil (Department of Neurosurgery, Duke University), Kara Baetz, Michelle Ouvina, Mollie Witzel (Institute for Behavioral Medicine)

Abstract:
Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may persist years after injury. Aged individuals are especially vulnerable to fall-related TBI and account for the majority of TBI-related hospitalizations and deaths. The neurobiological mechanisms of aging that support worse outcomes after TBI are undefined. Our main objective was to compare the neuroinflammatory response to diffuse TBI between adult and aged mice to elucidate mechanisms of aging that confer risk of worse outcomes. Here, adult (2 mo) and aged (16-18 mo) C57BL/6 mice were subjected to a mild diffuse brain injury, induced by midline fluid percussion, after which several biochemical and behavioral parameters were assessed 7 days post injury (dpi). Acute cognitive impairment was evident in both adult and aged TBI mice. There was enhanced reactive morphology of microglia and astrocytes in the cortex and hippocampus of aged mice compared to adults. Neuropathology mRNA analysis showed amplified cytokine/chemokine, complement, innate immune, and interferon-associated gene expression in the cortex of aged mice after TBI compared to adults. Ingenuity Pathway Analysis indicated mediators of interferon (IFN) signaling as robust upstream regulators of genes enhanced with age after TBI. Based on these results, we used DMXAA, a STING (stimulator of interferon genes) agonist, to determine if enhanced IFN signaling would worsen neuroinflammation after TBI in adult mice. Adult mice administered DMXAA after TBI had amplified expression of myriad genes that were amplified in Aged-TBI mice. Overall, IFN signaling induced by TBI is critical in the transition from acute to chronic neuroinflammation, and activation of this pathway is especially prominent in aged mice.

Keywords: Aging, Traumatic Brain Injury, Interferons

68. The role of SRP54 in motor neuron development and implications for SMA

Nikaela Losievski (Department of Neuroscience), Amy Everest, PhD (Department of Neuroscience and Department of Molecular Genetics, The Ohio State University, Columbus, OH), Thomas L. Gallagher, PhD (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Alan Kessler, PhD, Christine E. Beattie, PhD (Department of Neuroscience, The Ohio State University, Columbus, OH), Sharon L. Amacher, PhD (Department of Molecular Genetics and Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH), Stephen J. Kolb, MD, PhD (Department of Biological Chemistry and Pharmacology and Department of Neurology, The Ohio State University, Columbus, OH)

Abstract:
Spinal muscular atrophy (SMA) is a motor neuron disease typically caused by a mutation in or deletion of the SMN1 gene which reduces the amount of survival of motor neuron (SMN) protein throughout an organism. All cells require SMN to survive, but the first observed symptoms in SMA patients are motor neuron dysfunction and death. It is unknown why motor neurons are especially sensitive to deficient functional SMN protein. SMN oligomerizes to form the SMN complex whose canonical function is small nuclear ribonucleoprotein (snRNP) assembly. Current research fails to demonstrate how dysfunctional snRNP assembly impacts motor neurons more severely than other tissue types. Other proposed functions for the SMN complex may explain tissue specificity in SMA. In zebrafish, tagged SMN protein was expressed under a motor neuron-specific promoter to explore SMN associations in motor neurons. One SMN-associated protein, Srp54, is a component of the signal recognition particle (SRP), an RNP responsible for processing a subset of secreted and integral membrane proteins. The SMN complex had previously been implicated in SRP assembly. If the SMN:SRP54 interaction is critical for motor neuron health, we predicted that srp54-/- zebrafish would have motor axon defects similar to smn-/-smn-/- zebrafish. We obtained a srp54-/- zebrafish line with a premature stop codon predicted to either encode a severely truncated protein or result in nonsense mediated decay of the transcript. Immunohistochemistry and confocal imaging revealed that srp54-/- embryos have truncated and/or aberrantly branched motor axons, reminiscent of the smn-/- embryos. The similar motor axon defects in the srp54-/- and smn-/- embryos support our hypothesis that an SMN:SRP association may be particularly important in motor neurons. This work will further the understanding of motor neuron selectivity in SMA and may provide insight into other motor neuron diseases.

Keywords: Spinal muscular atrophy, Motor neurons, Signal recognition particle

69. Deep Learning-Based Identification of Microglial Cellular Populations in Glioblastoma Patients

Wesley Wang (Neuroscience Graduate Program, The Ohio State University), Jonah D. Tugaoen (Department of Pathology, The Ohio State University), Jose J. Otero (Department of Pathology, The Ohio State University)

Abstract not available online - please check the booklet.

70. Hepatic sympathetic denervation improves intraspinal tissue sparing after spinal cord injury

Anthony N. Alfredo (Neuroscience Graduate Program), Dana M. McTigue (Department of Neuroscience, The Ohio State University)

Abstract:
Spinal cord injury (SCI) is a devastating neurological impairment affecting locomotor function and physiological homeostasis. There are approximately 250 – 375,000 individuals living with SCI in the US, and average life expectancy post-injury has not improved in nearly 40 years. Because the spinal cord innervates all the organs of the body, SCI causes detrimental impairments of visceral organs and their regulation. Recent studies from our group reveal that the liver plays a role in the inflammatory response after SCI and that liver inflammation at the time of SCI exacerbates spinal cord pathology. Additionally, we determined lipid accumulation and liver inflammation persist chronically and lead to a novel form of neurogenic non-alcoholic steatohepatitis (nNASH). However, the mechanism of post-SCI nNASH is currently not known. Reports in the literature suggest that the sympathetic nervous system (SNS) activates Kupffer cells (KCs), the resident macrophages of the liver, increasing liver inflammation and facilitating the development of NASH. Therefore, we hypothesize that removing sympathetic input to the liver following SCI will reduce liver and spinal cord pathology. Thus, rats received a moderate contusion injury at T8. At 14 days post-injury (dpi), injured rats received a hepatic sympathectomy (hSymX) or sham surgery to remove SNS innervation specifically to the liver. Animals survived for 14d post-hSymX and were sacrificed at 28 dpi by intracardiac perfusion. In contrast to our hypothesis, liver histology revealed a surprising significant increase in KCs in animals receiving hSymX + SCI compared to hSymX Sham + SCI. However, hSymX + SCI animals had significantly more spared white matter and decreased macrophages in the spinal cord compared to hSymX Sham + SCI. Thus, SNS innervation to the liver plays a complicated role in SCI recovery. Future studies will investigate the mechanism of action for increased spinal tissue sparing with concomitant increased KC activation after post-SCI removal of liver SNS innervation to determine if the neuroprotective effects can be preserved without enhancing hepatic KC activation.

Keywords: Spinal Cord Injury, Inflammation, Kupffer Cells

71. Molecular mechanisms underlying enhancement of the tripartite glutamatergic synapse by LH001, a clinical candidate compound for Alzheimer’s disease

Zan Xu (OSU neuroscience graduate program), Joshua Foster (OSU department of neuroscience), Xueqin Wang (OSU department of neuroscience), Chien-liang Lin (OSU department of neuroscience)

Abstract:
Tripartite synapse is formed by a pre-synapse, a post-synapse, and a peri-synaptic astrocytic process (PAP) (1). Studies indicate that loss of tripartite glutamatergic synapses is the major correlate of cognitive impairment in Alzheimer’s disease (AD) (1). Restoring tripartite glutamatergic synapse is a potential therapeutic strategy for AD (1). Our laboratory has discovered and developed a novel small molecule named LH001 that can enhance the structure and function of tripartite glutamatergic synapses (2). LH001 can effectively restore tripartite glutamatergic synapses and significantly improve cognitive functions in two mouse models of AD (2,3). However, the molecular mechanisms of LH001 remain to be fully elucidated. In a previous study, we found that LH001 action site is located at the PAP. A set of PAP proteins is rapidly upregulated through local translation following LH001 treatment. Recently, we found that a set of proteins localized to the plasma membrane of the PAP immediately after LH001 treatment. LH001 mediated localization was abolished by inhibition of protein kinase A (PKA), indicating PKA signaling pathway involvement. In addition, subunits of eIF4F complex were found localized to the plasma membrane and were activated by PKA after LH001 treatment. In light of these observations, we hypothesize that LH001 activates PKA signaling pathway resulting in localization and activation of proteins involved in translation at the plasma membrane of the PAP, which increases protein synthesis. To test this hypothesis, we used BONCAT (bio-orthogonal non-canonical amino acid tagging) to label newly synthesized proteins in primary astrocyte cultures. The result showed that LH001 increased protein translation, and this effect was abolished by a PKA inhibitor. We currently i) investigate if LH001 mediated protein translation occurs at the plasma membrane of the PAP ii) identify these newly synthesized proteins, and iii) examine the effects of protein translation at the PAP on the functional and structural plasticity of the tripartite synapses.

References:
1. Rudy CC, Hunsberger HC, Weitzner DS, Reed MN. The Role of the Tripartite Glutamatergic Synapse in the Pathophysiology of Alzheimer’s Disease. Aging Dis. 2015 Mar 10;6(2):131–48.
2. Foster JB, Zhao F, Wang X, Xu Z, Lin K, Askwith CC, et al. Pyridazine-derivatives Enhance Structural and Functional Plasticity of Tripartite Synapse Via Activation of Local Translation in Astrocytic Processes. Neuroscience. 2018 Sep;388:224–38.
3. Takahashi K, Kong Q, Lin Y, Stouffer N, Schulte DA, Lai L, et al. Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer’s disease. J Exp Med. 2015 Mar 9;212(3):319–32.

Keywords: Tripartite synapse, Alzheimers disease, protein translation

72. Neuron selective tagging, tracing, and capture reveals unique pattern of activation within the prefrontal cortex and hippocampus following social stress

Rebecca Biltz (Neuroscience Graduate Program), Wenyuan Yin (Neuroscience Graduate Program), Braedan Oliver (Institute for Behavioral Medicine Research), John Sheridan (Division of Biosciences, Institute for Behavioral Medicine Research), Jonathan Godbout (Department of Neuroscience, Institute for Behavioral Medicine Research)

Abstract not available online - please check the booklet.

73. Microglia are implicated in brain region-specific tumor-induced neuroinflammation and behavioral comorbidities

Lindsay D. Strehle (Neuroscience Graduate Program; Institute for Behavioral Medicine Research), Corena V. Grant (Institute for Behavioral Medicine Research), Lauren Otto (Institute for Behavioral Medicine Research), Leah M. Pyter (Neuroscience; Psychiatry and Behavioral Health; Institute for Behavioral Medicine Research)

Abstract:
Breast cancer is the most common cancer among females worldwide. Following diagnosis, up to 30% of these patients report mood disturbances (i.e., anxiety, depression). Our tumor-bearing rodent model recapitulates the elevation of circulating inflammatory markers observed in cancer populations and displays concurrent increased proinflammatory mediators in the brain and negative affective-like behaviors. Neuroinflammation is a proposed mechanism behind the etiology of mood disorders and therefore may be contributing to these mood disturbances. However, the cellular mechanisms by which tumor-induced neuroinflammation occur remain unknown. We hypothesize that microglia are the primary cells driving tumor-induced neuroinflammation and therefore affective-like behaviors. We used young adult female Balb/c mice, half of which were induced with an orthotopic, syngeneic, non-metastatic 67NR mammary tumor; the other half underwent a sham surgery. Experiment 1 was designed to assess brain region-specific morphological (IHC) and gene expression activation of microglial cells (Ccl2, Il1b, Il6, Tnfa). Experiment 2 was designed to test the necessity of microglia in tumor-induced behavioral and neuroinflammatory outcomes using a colony stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) via chow. Remarkably, tumors did not alter the expression of primary microglia genes investigated relative to controls from Experiment 1. In the open field test of Experiment 2, tumor-bearing mice unexpectedly displayed greater entries and increased locomotion in the center of the field than tumor-free mice; these tumor-induced risky behaviors were attenuated by PLX5622 chow. Microglial depletion reversed tumor-induced increases in Il6 gene expression in the hippocampus without reducing peripheral inflammatory markers. These preliminary results suggest that microglia may have region- and mediator-specific involvement in tumor-induced neuroinflammation. IHC analyses are currently underway. This research will advance our understanding of the mechanisms underlying tumor-induced neuroinflammation and the associated behavioral deficits in order to identify specific cellular and/or molecular targets to mitigate cancer-associated behavioral comorbidities.

Keywords: mammary tumor, neuroinflammation, behavior

74. Neuronal cilia modulate D1 mediated dopaminergic signaling

Toneisha Stubbs (Neuroscience Graduate Program ), Kirk Mykytyn (Department of Pharmacology ), Candice Askwith (Department of Neuroscience)

Abstract:
Primary cilia are rod shaped cellular appendages found on nearly every cell type including most central neurons. Dysfunction in the primary cilium is implicated in a class of human disorders termed ciliopathies. Ciliopathies are associated with numerous neuropathologies, including cognitive deficits, obesity, and behavioral phenotypes. Important insight into the function of neuronal cilia in the mammalian brain came from the finding that neuronal cilia are enriched for certain G protein-coupled receptors (GPCRs) and downstream effectors of GPCR signaling, suggesting that neuronal cilia respond to neuromodulators in the extracellular environment. We have previously shown that GPCR ciliary localization is disrupted in neurons from mouse models of the human ciliopathy Bardet-Biedl syndrome (BBS). Interestingly, we found that while certain GPCRs failed to localize to neuronal cilia in BBS mouse models the GPCR dopamine receptor 1 (D1) accumulated in neuronal cilia. These findings led to us to hypothesize that ciliary GPCR mislocalization impacts receptor signaling and contributes to BBS phenotypes such as obesity. Our work shows that disrupting the ciliary localization of D1 by either loss of a BBS protein or lack of cilia, on D1 expressing neurons leads to decreased D1 mediated dopaminergic signaling. Additionally, we observed that ciliary mislocalization of D1 also resulted in obesity. Further investigation showed that this increase in weight was not due to increased food intake but was associated with decreased locomotor activity. Taken all together these observations show that proper cilia formation and function are required for normal dopaminergic signaling in D1 expressing neurons. To further explore the impact that neuronal cilia have on D1 expressing neurons future directions and ongoing work aims to 1) identify the proteins involved in trafficking D1 to the restricted ciliary membrane and 2) identify the downstream transcriptional consequences of D1 ciliary mislocalization on D1 expressing neurons.

References:
1. Domire, J. S., Green, J. A., Lee, K. G., Johnson, A. D., Askwith, C. C., & Mykytyn, K. (2010). Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the bardet-biedl syndrome proteins. Cellular and Molecular Life Sciences : CMLS, 68(17), 2951-2960. doi:10.1007/s00018-010-0603-4
2. Berbari, N. F., Lewis, J. S., Bishop, G. A., Askwith, C. C., & Mykytyn, K. (2008). Bardet Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proceedings of the National Academy of Sciences - PNAS, 105(11), 4242-4246. doi:10.1073/pnas.0711027105
3. Hilgendorf, K. I., Johnson, C. T., & Jackson, P. K. (2016). The primary cilium as a cellular receiver: Organizing ciliary GPCR signaling. Current Opinion in Cell Biology, 39, 84-92. doi:10.1016/j.ceb.2016.02.008

Keywords: Dopamine Receptor 1, Neuronal Primary Cilia, Bardet Biedl Syndrome

75. Social isolation worsens diet-induced obesity and alters metabolic and inflammatory markers in the hypothalamus

Jacqueline M. Anderson (Neuroscience Graduate Program), Nicholas J. Queen, Rhiannon Bates, Wei Huang, Suraj Komatineni (Department of Cancer Biology & Genetics, College of Medicine, The Ohio State University), Xiaokui Mo (Department of Biomedical Informatics, College of Medicine, The Ohio State University), Anthony G. Mansour, Run Xiao, Logan A. Chrislip, Lei Cao (Department of Cancer Biology & Genetics, College of Medicine, The Ohio State University)

Abstract:
Social isolation (SI) is a risk factor for a variety of chronic diseases, including obesity and metabolic syndromes. Disruptions in hypothalamic function have been implicated in both social isolation and obesity, but underlying hypothalamic mechanisms for how social isolation may affect the development of obesity have not been extensively researched. Additionally, the majority of murine studies are performed under room-temperature conditions. Mice that are singly housed are unable to socially thermoregulate, thereby potentially exacerbating chronic cold stress and masking the effects of psychosocial stress. Here, we placed SI-housed and group-housed mice under thermoneutral (TN) conditions and fed them a high-fat diet (HFD) to determine the impact of SI on the development of obesity and on the hypothalamus. We found that SI worsened metabolic outcomes and altered metabolic and inflammatory hypothalamic markers, including downregulation of Bdnf and Mc4r expression. Interestingly, we found that SI downregulated several genes involved in the neuroinflammatory response. In a subset of normal chow-fed mice exposed to acute SI, we subjected the hypothalamus and amygdala to a microarray and transcriptomic analysis to examine early brain changes in response to SI. Acute SI altered the transcriptome of the hypothalamus and amygdala, with Hdc and Fos being highly induced by SI. Current experiments are underway to further investigate the hypothalamic inflammatory response to SI and HFD as well as experiments to explore the involvement of HDC in the SI phenotype.

Keywords: social isolation, obesity, hypothalamus

76. Unraveling molecular and cellular mechanisms in a mouse model of SCN8A developmental and epileptic encephalopathy

Midhun N. K. Anne (MCDB), Jason Kaplan (Department of Neuroscience), Laura Kakuk-Atkins (Department of Neuroscience), Jacy L. Wagnon (Department of Neuroscience)

Abstract:
Developmental and epileptic encephalopathies (DEEs) are genetic disorders in humans characterized by drug-resistant seizures along with developmental delay, motor impairment, and neurological deficits. Genome sequencing has identified pathogenic variants in voltage-gated sodium channel alpha subunit genes, including SCN1A and SCN8A, as prominent causes of DEE. The SCN8A gene encodes Nav1.6 protein, which is a pore-forming alpha-subunit of voltage-gated sodium channels that is localized to the axon-initial segments of neurons to initiate and propagate action potentials. A previous mouse model with the patient mutation Scn8a-N1768D exhibited early-seizure onset but did not recapitulate developmental delay and motor impairment seen in the patient. We have developed a conditional mouse model of SCN8A encephalopathy based on a patient mutation that cause severe neonatal-onset seizures and developmental delay. The Scn8a-T767I mouse recapitulates early-onset seizures similar to the proband. Strikingly, Scn8a-T767I mice also exhibit developmental delay and motor impairment along with severely reduced life span. Compared to WT mice, Scn8a-T767I mice showed severe motor impairment demonstrated by delayed or non-existent righting reflex and deficits in several other motor coordination tests. Using neuron type specific-Cre lines and in-vitro studies, our new mouse model will help in understanding the molecular and cellular changes that contribute to developmental delay, epileptogenesis and comorbidities in SCN8A-related DEE.

References:
1. Specchio N, Curatolo P. Developmental and epileptic encephalopathies: what we do and do not know. Brain. 2021 Feb 12;144(1):32-43.
2. Bender AC, Morse RP, Scott RC, Holmes GL, Lenck-Santini PP. SCN1A mutations in Dravet syndrome: impact of interneuron dysfunction on neural networks and cognitive outcome. Epilepsy Behav. 2012;23(3):177-186.
3. Veeramah KR, O'Brien JE, Meisler MH, et al. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet. 2012;90(3):502-510.
4. Estacion M, O'Brien JE, Conravey A, et al. A novel de novo mutation of SCN8A (Nav1.6) with enhanced channel activation in a child with epileptic encephalopathy. Neurobiol Dis. 2014;69:117-123.

Keywords: Voltage-gated sodium channel, Epilepsy, Developmental Delay

77. Characterizing the potential dysbiosis in gut viral and microbial communities following Spinal Cord Injury

Mohamed Mohssen (The Interdisciplinary Biophysics Graduate Program, The Ohio State University, Columbus, OH 43210, USA), Ahmed A. Zayed (Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA), Kristina A. Kigerl (Department of Neuroscience, The Ohio State University College of Medicine, Columbus, Ohio, USA), Phillip G. Popovich (Department of Neuroscience, The Ohio State University College of Medicine, Columbus, Ohio, USA), Matthew B. Sullivan (Department of Microbiology, The Ohio State University, Columbus, Ohio, USA)

Abstract not available online - please check the booklet.

78. Uncovering computational principles for thermoregulation in ectotherms through behavioral characterization in larval zebrafish

Kaarthik Balakrishnan (Biophysics Graduate Program, The Ohio State University), Martin Haesemeyer (Assistant Professor, Department of Neuroscience, The Ohio State University)

Abstract:
Avoidance of extreme temperatures is critical for all living organisms. Endotherms use internal and behavioral mechanisms to regulate their body temperature. Ectotherms on the other hand are in equilibrium with their surroundings and rely on behavioral mechanisms to thermoregulate by navigating thermal profiles in the environment. Ectotherms are therefore ideally suited to provide insight into the relation between thermosensory input and behavioral output. Larval zebrafish are excellent model organisms to study mechanisms of behavioral thermoregulation because they are ectotherms with an archetypical vertebrate brain. Furthermore, their behavioral repertoire is highly tractable, consisting primarily of modulating the direction of motion and swim speeds. Our previous work uncovered how larval zebrafish avoid hot temperatures by raising their swim speeds in warmer water and performing evasive turns in the presence of increasing temperatures. However, it is critical to understand behavioral responses to both hot and cold temperatures in order to develop a model that describes thermoregulation of an organism, and to specify a notion of preferred temperature regime for the organism. Hence, we will characterize the behavior of freely swimming larval zebrafish in cold temperatures by tracking their movement in a chamber with temperature gradients. We will develop a model of thermoregulation by relating absolute temperatures and changes in temperature to swim speeds and turns, in a range of hot and cold temperatures. This model will reveal the underlying computational principles which transform external temperature input to behavioral output and its relation to the position of the organism in temperature space. Subsequently, this will inform future investigations of possible neural circuits giving rise to thermoregulatory behavior and the encoding of temperature preference in the brain.

References:
1. Haesemeyer M, Robson DN, Li JM, Schier AF, Engert F. A Brain-wide Circuit Model of Heat-Evoked Swimming Behavior in Larval Zebrafish. Neuron. 2018;98(4):817-831.e6. doi:10.1016/j.neuron.2018.04.013
2. Luo L, Gershow M, Rosenzweig M, et al. Navigational decision making in Drosophila thermotaxis. J Neurosci. 2010;30(12):4261-4272. doi:10.1523/JNEUROSCI.4090-09.2010

Keywords: Thermoregulation, Larval zebrafish, Computational modeling

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

Emma Woodward (Neuroscience Graduate Program), Laurence Coutellier (Department of Psychology, The Ohio State University)

Abstract not available online - please check the booklet.

80. 5’ splice-site variants in KIF5A selectively result in exon 27 exclusion, contributing to ALS

Megan G. Pino, MS (Neuroscience Graduate Program), Nicholas J. Hall (Neurology), Chathuri Pathirage (Chemistry), Karin Musier-Forsyth, PhD (Chemistry), Arthur Burghes, PhD (Biological Chemistry and Pharmacology, Molecular Genetics), Stephen J. Kolb, MD, PhD (Neurology, Biological Chemistry and Pharmacology)

Abstract:
Kinesin Family Member 5A (KIF5A) is a gene that encodes one of the heavy chain regions of kinesin-1, a motor protein involved in cellular transport along microtubules.1 Single nucleotide variants in the cargo-binding domain of KIF5A have been linked to amyotrophic lateral sclerosis (ALS), a rapidly progressive and fatal neuromuscular disease that primarily affects motor neurons.2-5 However, the mechanisms by which KIF5A variants contribute to ALS remain unknown. Approximately one-third of all disease-associated genetic mutations exhibit disruption of the correct pattern of pre-mRNA splicing,6,7 and 12 of the 14 KIF5A variants identified are located near exon 27 splice-site junctions. Thus, we hypothesized that mis-splicing of the penultimate exon 27 results in aberrant KIF5A RNA and protein, resulting in a novel C-terminus that gains a toxic function and causes ALS. To test this, we developed an in vitro splicing assay to detect full-length (exon 27 inclusion) and mis-spliced (exon 27 exclusion) KIF5A mRNA isoforms. We performed ddPCR in multiplexed reactions with a normalizer assay to quantify expression and calculate the rate of exon 27 inclusion for each KIF5A variant. First in HEK 293 cells transfected with KIF5A variant plasmids, we showed that 5' splice-site (5'ss) variants in KIF5A selectively result in exon 27 exclusion from mRNA. We then confirmed this result in CRISPR-Cas9 genetically edited human motor neurons, a disease-relevant cell type. Further, in an in vivo mouse model of one 5'ss variant, we observed a decrease in Kif5a protein expression in addition to RNA mis-splicing, implicating disrupted transcription and translation in KIF5A-linked ALS pathogenesis. We believe this is due to disruption of consensus 5'ss sequences where crucial ribonuclear proteins (RNPs) - such as U1 RNP - bind. We are now performing Western blot quantification and pulse-chase experiments to quantify KIF5A protein levels and the rate of protein degradation, respectively. In parallel, we are performing in vitro RNA structure probing utilizing SHAPE-MaP to generate secondary structures at single-nucleotide resolution and identify essential splicing domains that could potentially be targeted with RNA-based therapeutics.8,9 We will present updated findings at the symposium.

References:
1. NCBI KIF5A Gene. https://www.ncbi.nlm.nih.gov/gene/3798
2. NINDS ALS Fact Sheet https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Amyotrophic-Lateral-Sclerosis-ALS-Fact-Sheet
3. Brenner D et al. 2018. https://doi.org/10.1093/brain/awx370
4. Nicolas A et al. 2018. https://doi.org/10.1016/j.neuron.2018.02.027
5. He J et al. 2020. https://doi.org/10.1136/jnnp-2019-320483
6. Cáceres JF & Kornblihtt AR 2002. https://doi.org/10.1016/s0168-9525(01)02626-9
7. Montes M et al. 2019. https://doi.org/10.1016/j.tig.2018.10.002
8. Merino EJ et al. 2005. https://doi.org/10.1021/ja043822v
9. Smola MJ et al. 2015. https://doi.org/10.1038/nprot.2015.103

Keywords: Kinesin Family Member 5A (KIF5A), Amyotrophic lateral sclerosis (ALS), RNA splicing and processing mechanisms

81. Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by mediating immunothrombosis

Mostafa Eltobgy (Neuroscience Graduate Program), Ashley Zani (Department of Microbial infection and immunity), Andrea Tedeschi (Department of Neuroscience), Shahid Nimjee (Department of Neurosurgery), Jacob Yount (Department of Microbial infection and immunity), Amal Amer (Department of Microbial infection and immunity)

Abstract:
SARS-CoV-2 infections are a worldwide health concern, and new treatment strategies are needed for decreasing virus-induced inflammatory tissue damage. Severe inflammatory response and coagulopathy are the hallmarks of severe Covid-19. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive.
Here, we show that the innate immunity proteins, human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice experienced less severe infections in terms of weight loss and lung damage than WT mice. Notably, these phenotypes were not recapitulated in mice lacking the CASP11 downstream effector gasdermin D (Gsdmd-/-), though viral titers were similar in all groups. Global transcriptomics of infected WT and Casp11-/- lungs identified decreased inflammation and neutrophil gene signatures. We confirmed that protein levels of inflammatory mediators IL-1β and CXCL1, and neutrophil infiltration, were decreased in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial injury/activation, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity.
These findings establish CASP11 as an upstream regulator endothelial cell dysfunction and coagulopathy in SARS-CoV-2 infection largely independently of GSDMD. Overall, our results demonstrate that CASP11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, identifying CASP11 as a promising drug target for treatment and prevention of tissue damage in COVID-19.

Keywords: SRAS-CoV-2, Immunothrombosis, Caspase4

82. Adaptive changes in viral envelope resulting from adaptation of simian-tropic HIV-1 to macaques confers resistance to interferon

Anna C. Smith (Molecular, Cellular, and Developmental Biology), Haidyn Weight (Human Biology Division, Fred Hutchinson Cancer Research Center), Julie Overbaugh (Human Biology Division, Fred Hutchinson Cancer Research Center), Amit Sharma (Veterinary Biosciences, Microbial Infection and Immunity)

Abstract not available online - please check the booklet.

83. SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling

Tai-Wei Li (Department of Pathology, OSUMC), Adam D. Kenney (Department of Microbial Infection and Immunity, OSUMC), Jun-Gyu Park (Texas Biomedical Research Institute, San Antonio, TX ), Jianwen Que (Department of Medicine, Columbia University Medical Center, New York, ), Luis Martinez-Sobrido (Texas Biomedical Research Institute, San Antonio, TX ), Jacob S. Yount (Department of Microbial Infection and Immunity, OSUMC)

Abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMDPH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

References:
Zaffagni M, Harris JM, Patop IL, Pamudurti NR, Nguyen S, Kadener S. 2022. SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome. Elife 11.

Keywords: SARS-CoV-2, Nsp14, NF-kB

84. Generation of a live attenuated RSV vaccine with optimized immunogenicity

Samiha Kabir (MCDB)

Abstract:
Human respiratory syncytial virus (RSV) infects all children by the age of two. RSV causes severe infection in immunocompromised children and adults. It is deemed a major cause of infant mortality and is a significant burden on the economy. There is no approved vaccine for RSV. RSV has two subtypes, A and B, which circulate in dominance. Antibodies against one subtype can neutralize the other, though not as efficiently. We propose to generate a live attenuated vaccine (LAV) candidate expressing two fusion (F) proteins from two different subtypes. The fusion protein of RSV is critical for establishing successful infection and induces the highest titer of neutralizing antibodies (nAb). The pre fusion (pre-F) form of F retains the two most immunogenic sites of the protein and is the most effective antigen for eliciting nAb. Pre-F will be stabilized and will be incorporated into our LAV. Furthermore, we will test the efficacy of our LAV in vivo by immunizing cotton rats followed by wt RSV challenge. Together, these strategies will generate an LAV with a broader, enhanced immune response that will protect against both RSV A and B.

Keywords: RSV, LAV

85. Neutralizing antibody responses elicited by SARS-CoV-2 mRNA vaccination wane over time and are boosted by breakthrough infection

John P. Evans (Molecular, Cellular, and Developmental Biology), Cong Zeng (Department of Veterinary Biosciences, The Ohio State University), Claire Carlin, Joseph S. Bednash, Rama Mallampalli, Richard J. Gumina (Department of Internal Medicine, The Ohio State University), Gerard Lozanski (Department of Pathology, The Ohio State University), Eugene M. Oltz (Department of Microbial Infection and Immunity), Linda J. Saif, Shan-Lu Liu (Viruses and Emerging Pathogens Program, Infectious Disease Institute, The Ohio State Unversity)

Abstract:
SARS-CoV-2 is the causative agent of the ongoing COVID-19 pandemic which has resulted in nearly 400 million cases and over 5 million deaths. Unprecedented efforts from the scientific community led to the development of two mRNA vaccines, among others, that have helped to control the worst impacts of the pandemic. However, the durability of protection for these vaccines and how they compare to immunity from natural infection, remain critical concerns. Additionally, the emergence of SARS-CoV-2 variants of concern (VOCs) which exhibit resistance to vaccine-induced neutralizing antibodies (nAbs), continue to threaten the efficacy of the mRNA vaccines. To address these concerns we developed a highly sensitive pseudotyped lentivirus-base virus neutralization assay to examine nAb titers in COVID-19 patients and mRNA vaccine recipients. With this we demonstrate that mRNA vaccinated health care workers (HCWs) reliably exhibit strong nAb response. However, the VOCs exhibit varying degrees of nAb escape with the Omicron variant exhibiting near complete escape from 2-dose mRNA vaccine-induced nAbs, and 2-dose induced nAbs wane substantially 6 months after vaccination against all VOCs. Following booster vaccination 83.3% of HCWs exhibited detectable nAb titers against Omicron, indicating a need for booster vaccine administration for strong protection from Omicron. Additional examination of sera from Delta and Omicron COVID-19 patients demonstrated strong resistance of Omicron to Delta patient sera but sensitivity to Omicron patient sera. Omicron variant sub-lineages were also examined to discern any differences in sensitivity to mRNA-vaccine- or Omicron-infection-induced nAbs. Overall, our results serve to inform the administration of booster doses and any need for a reformulation of mRNA vaccines.

References:
Evans, J.P., Zeng, C., Carlin, C., Lozanski, G., Saif, L.J., Oltz, E.M., Gumina, R.J. and Liu, S.L., 2022. Neutralizing antibody responses elicited by SARS-CoV-2 mRNA vaccination wane over time and are boosted by breakthrough infection. Science Translational Medicine, p.eabn8057.

Keywords: SARS-CoV-2, Neutralizing Antibody, Variants of Concern

86. Investigating the role Of endothelial cell-expressed Jag1 on smooth muscle function and vascular homeostasis

Randa Breikaa (MCDB)

Abstract not available online - please check the booklet.

87. Mineralocorticoid receptor signaling in the microenvironment of dystrophic skeletal muscles

Chetan K. Gomatam (Molecular, Cellular, and Developmental Biology Program, The Ohio State University), Zachary M. Howard (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University), Pratham Ingale (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University), Jeovanna Lowe (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University), Shyam S. Bansal (Department of Physiology and Cell Biology and Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University), Jill A. Rafael-Fortney (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University)

Abstract:
Duchenne muscular dystrophy (DMD) is a fatal disease resulting from the absence of dystrophin that causes chronic striated muscle injury and gradual muscle degeneration, leading to destruction of muscle and loss of life in the mid-twenties. Chronic injury occurs due to cycles of muscle degeneration and regeneration, which leads to overactivation of fibroblasts. Fibroblasts are normally essential for organ structure and wound healing via their ability to temporarily stabilize injury sites by depositing extracellular matrix (ECM) components. However, fibroblasts cause pathogenic fibrosis when overactivated due to excess ECM deposition that replaces muscle tissue in DMD. We have identified mineralocorticoid receptor (MR) antagonists as a potential therapy to alleviate fibrosis and inflammation in muscular dystrophies. However, effects of MR signaling on fibroblasts and the role of MR in fibroblasts are not known.
Using fluorescence activated cell sorting and gene expression analyses in MR antagonist-treated dystrophic mdx mice and in mdx mice with cell-specific MR conditional knockouts (MRcko), we have begun exploring the fibroblast response to MR signaling in dystrophic muscle and the role of fibroblast-specific MR. Our recent gene expression microarray of MRcko-mdx mice and RNA sequencing of mdx myeloid immune cells from dystrophic muscles suggest that there is significant crosstalk between immune cells, fibroblasts, and myofibers in DMD. Several genes involved in regulation of ECM and development of fibrosis were significantly upregulated in myeloid cell-specific MRcko-mdx mice versus myofiber-specific MRcko-mdx mice, including fibronectin, tenascin, and lysyl oxidase. In vivo and in vitro assays combined with genetic approaches will be used to decipher whether MR signaling in the muscle microenvironment has indirect and direct effects on fibroblast activation and function, and whether MR signaling differs between muscle groups and between chronic and acute injury.

Keywords: Muscular dystrophy, Fibrosis, Mineralocorticoid receptor

88. Regulation of AKT signaling dynamics by the IGF-IGFBP axis in the lung metastatic microenvironment

Rawan Makkawi (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Vaibhav Murthy (Department of Veterinary Biosciences, The Ohio State University), Alexander Davies (Department of Veterinary Biosciences, The Ohio State University)

Abstract:
The lung is a common metastasis site for various cancers, such as triple negative breast cancer. During metastasis, disseminated cancer cells are able to adapt to the signaling cues in the lung microenvironment, resulting in changes in their signaling pathways and their responsiveness to chemotherapeutics. Of particular interest is the PI3K/AKT pathway, which regulates cell growth and proliferation. It is dysregulated in a variety of human cancers, making it a potential target for therapy. AKT signaling is highly sensitive to Insulin-like growth factor (IGF) and its cognate receptor, IGFR. One of the key regulators of IGF signaling are IGF-binding proteins (IGFBP), a family of proteins that interact with IGF and both positively and negatively modulate its function. Although the IGF-IGFBP signaling has been implicated in several cancers, how IGFBP production by cancer cells and cells of the microenvironment influence cancer cell signaling is yet to be studied in fine detail. We hypothesize that AKT signaling is regulated by the IGF-IGFBP axis in a manner that favors persistent activation of pro-oncogenic AKT signaling in cancer cells. Consistent with this hypothesis, our preliminary data show that changes in IGF levels, or drug treatments, initially suppress AKT signaling but is followed by restoration of AKT signaling in cancer cells to basal levels over time. These changes correlate with altered IGFBP expression profiles in cells of the lung microenvironment, suggesting an adaptive regulatory mechanism to compensate for low AKT signaling. Our future work aims to understand the regulation of cancer cell signaling more fully through IGF and IGFBPs in the metastatic microenvironment and develop strategies to suppress it.

Keywords: Metastatic Microenvironment, AKT Signaling

89. Developing DNA origami nanodevices from custom scaffolds to probe nucleosome arrays

Yin Wei (Biophysics Program), Blanche Chen (OSBP Program), Yuchen Wang (Department of Mechanical and Aerospace Engineering), Michael Poirier (Department of Physics), Carlos Castro (Department of Mechanical and Aerospace Engineering)

Abstract not available online - please check the booklet.

90. Characterization of a DNA Origami Force Probe

Ariel Robbins (Biophysics), Peter Beshay (Mechanical and Aerospace Engineering), Carlos Castro (Mechanical and Aerospace Engineering), Michael Poirier (Physics)

Abstract:
DNA origami nanotechnology is a rapidly developing field that shows promise in scientific applications such as mechanically aided drug delivery, molecular sensing, force sensing, and probing of single molecule dynamics. Complex and dynamic 3-dimensional structures can perform a prescribed function through controlled actuation making their use precise and reproducible. The device in our study, called a nanodyn, acts as a force sensor. Consisting of two origami bundles linked by six crossover strands, the device can exist in either an open or closed configuration. With careful design of the crossover strands, the nanodyn can be programed to open at a prescribed force. We will employ single molecule force spectroscopy techniques to characterize the force dependent properties of this device. These nanoprobes can then be used in biological systems where traditional force spectroscopy techniques are more challenging to implement. For instance, shear forces due to fluid flow can be challenging to determine in non-idealized environments, such as in a blood vessel or extracellular matrix. This device will supplement the existing force spectroscopy toolkit available to scientists for probing biological systems.

Keywords: DNA Origami, Force Spectroscopy, Nanotechnology

91. Title not available online - please see the booklet.

Michael Ryan-Simkins (OSBP)

Abstract not available online - please check the booklet.

92. Determine how pioneer transcription factors CBF1 recognizes and regulates nucleosome dynamics

Zhiyuan Meng (Biophysics Program), Priit Eek (Department of Biochemistry and Molecular Biology. Penn state university.)

Abstract:
Gene regulation is essential for cell proliferation and differentiation. In eukaryotic cells, many transcription factors (TFs), as well as transcription machinery, compete with nucleosomes to access their genomic targets. This process is facilitated by a special group of TFs called “pioneer factors” (PFs).[1] PFs can invade compact chromosomes[2], remodel nucleosomes near their binding sites, direct the binding of other TFs, and trigger the transcription. Despite their essential functions, we lack understanding of the fundamental principles that govern the activity and function of PFs. The overall goal of my research is to try to answer the questions: 1) how do pioneer transcription factor recognize their binding sites inside the compact nucleosomes to facilitate the chromatin opening? 2) What differentiates the PF compared to TF to make it has higher affinity to the nucleosome? 3) How do possible regulate the PFs and TFs? Multiply biophysical methods including ensemble fluorescence resonance energy Transfer(FRET), single molecular FRET, electrophoretic mobility shift assay (EMSA) experiments will be applied into the research.

References:
[1] Zaret KS, Lerner J, Iwafuchi-Doi M. Chromatin Scanning by Dynamic Binding of Pioneer Factors. Mol Cell 2016:665–7.
[2] Zhou B-R, Jiang J, Feng H, Ghirlando R, Xiao TS, Bai Y. Structural Mechanisms of Nucleosome Recognition by Linker Histones. Mol Cell 2015;59:628–38.

Keywords: Pioneer Factors, FRET, chromatin

93. Evidence for an acetylation-independent function of the SAGA and ATAC human acetyltransferase modules

Kristin Chesnutt (OSBP, Ohio State University), Khan Cox (Department of Physics, Ohio State University), Christine Toelzer (School of Biochemistry, University of Bristol), Michael G. Poirier (Department of Physics, Ohio State University)

Abstract:
Post translational modifications (PTMs) play a crucial role in transcription regulation by recruiting regulatory complexes and by assisting in the modulation of DNA accessibility to transcription factors (TFs). Human Spt-Ada-Gcn5-acetyltransferase (SAGA) and ADA-two-A-containing (ATAC) are two distinct multifunctional co-activator protein complexes that contain similar histone acetyltransferase (HAT) modules. SAGA and ATAC can be targeted to genomic loci through interactions with histone PTMs and TFs. The mechanism in which SAGA and ATAC function with TFs to target and acetylate nucleosomes remains unknown. Here we used Fluorescence Anisotropy (FA) to characterize human SAGA and ATAC HAT module interactions with nucleosomes. Förster resonance energy transfer (FRET) was used to investigate how SAGA and ATAC HAT modules function with a TF (Gal4-VP16 and -DBD) to target nucleosomes. Additionally, Western blot assays were carried out to determine the influence TFs have on the acetylation activity of SAGA and ATAC HAT modules. We find that HAT modules of SAGA and ATAC bind with high affinity to nucleosomes, independent of TFs. We also find that the SAGA HAT module can facilitate Gal4- VP16 and DBD invasion into nucleosomes, whereas the ATAC HAT module can facilitate VP16’s invasion into nucleosomes independent of acetylation. This data is evidence for a new function of SAGA and ATAC HAT modules, displaying an ability to facilitate TF binding to nucleosomes that is acetylation-independent.

Keywords: chromatin, histone acetyltransferase, DNA accessibility

94. Title not available online - please see the booklet.

Songyu Dong (OSBP), Nicholas Pinkerton (Department of Chemistry and Biochemistry, The Ohio State University), Elena Kudryashova (Department of Chemistry and Biochemistry, The Ohio State University), Dmitri S. Kudryashov (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

95. Understanding the function of the La protein in Trypanosoma brucei

G. Lankani Gunaratne (Ohio State Biochemistry Program), Ananth Casius (Department of Microbiology, The Ohio State University), Richard J. Maraia (Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health), Juan D. Alfonzo (Department of Microbiology, The Ohio State University)

Abstract:
La is a ubiquitous nuclear RNA binding protein found in almost all eukaryotes investigated to date. Although this conservation suggests that La participates in an important process, its role in eukaryotic cells is not completely clear. Trypanosoma brucei, the causative agent of African Trypanosomiasis in humans, encodes a ‘classical’ genuine La protein (TbLa) with a La motif closely followed by an RNA recognition motif (RRM1), an additional RRM (RRM2α) and a C terminal short basic motif (SBM) along with a nuclear localization signal (NLS). We have shown that TbLa is essential for the survival of T. brucei through knocking down endogenous La by RNA interference (RNAi). A synonymous codon swapped version of myc-tagged TbLa, which is impervious to RNAi, (TbLa Rec) has been stably expressed in T. brucei and rescues a growth defect caused by RNAi down-regulation of endogenous La. Immunofluorescence microscopy showed that TbLa Rec resides mainly in the nucleus and partly in the cytoplasm, similar to endogenous La. Stable expression of an NLS-deleted TbLa Rec (TbLa NLSΔRec) in La RNAi cells rescues the growth phenotype similar to TbLa Rec. Immunofluorescence microscopy coupled with treatment of cells with Leptomycin B confirmed that TbLa with the NLS deletion indeed leads to exclusively a cytoplasmic localization of TbLa, but by rescuing the growth defect of endogenous La, it leads to the conclusion that the essential function of La is not necessarily due to its nuclear localization. We have also constructed a series of myc-tagged recoded La mutants with each of the RRM, SBM, and the NLS deleted. Through in vivo expression of individual RRM-deleted La mutants in endogenous La RNAi cells, we show that whereas RRM1 is essential for function, the second putative RRM2α is dispensable. The latter raises questions about why RRM2α is maintained, other than for structural rather than functional reasons.

References:
Blewett, N. H.; Maraia, R. J. La Involvement in tRNA and Other RNA Processing Events Including Differences among Yeast and Other Eukaryotes. Biochim. Biophys. Acta - Gene Regul. Mech. 2018, 1861 (4), 361–372.
Maraia, R. J.; Mattijssen, S.; Cruz-Gallardo, I.; Conte, M. R. The La and Related RNA-Binding Proteins (LARPs): Structures, Functions, and Evolving Perspectives. Wiley Interdiscip. Rev. RNA 2017, 8 (6), 1–43.
Foldynová-Trantírková, S.; Paris, Z.; Sturm, N. R.; Campbell, D. A.; Lukeš, J. The Trypanosoma Brucei La Protein Is a Candidate Poly(U) Shield That Impacts Spliced Leader RNA Maturation and tRNA Intron Removal. Int. J. Parasitol. 2005, 35 (4), 359–366.
Marchetti, M. A.; Tschudi, C.; Kwon, H.; Wolin, S. L.; Ullu, E. Import of Proteins into the Trypanosome Nucleus and Their Distribution at Karyokinesis. J. Cell Sci. 2000, 906, 899–906.

Keywords: La protein, Trypanosoma brucei, RNA binding proteins

96. Bioorganometallic reactivity of nickel-substituted azurin as a model for acetyl coenzyme A synthase

Alina Yerbulekova (The Ohio State University), Anastasia C. Manesis (The Ohio State University), Jason Shearer (Trinity University), Hannah Shafaat (The Ohio State University)

Abstract not available online - please check the booklet.

97. Understanding the role of EGFL7 in HSC precursors during HSC specification in the AGM

Rakeb Tafesse (The Ohio State University), Chinmayee Goda (The Ohio State University), Rohan Kulkarni (The Ohio State University), Girish Rajgolikar (The Ohio State University), Malith Karunasiri (The Ohio State University), Adrienne Dorrance (The Ohio State University)

Abstract:
In the hematological transplantation field, novel methods are needed to expand and maintain functional hematopoietic stem cells (HSCs) to increase the number of transplantable HSCs and potentially improve outcomes for patients1,2. Further understanding of factors and pathways that are capable of regulating HSC formation and/or functions is a critical step towards achieving the end goal of having successful hematopoietic stem cell transplantation (HSCT). Our unpublished data show that the lack of epidermal growth factor like 7 (EGFL7), a predominantly endothelial cell secreted protein, in adult mice led to a significant decrease in HSC numbers. In addition, in vivo treatment of wild type (WT) mice with exogenous EGFL7 or recombinant EGFL7 (rEGFL7) resulted in a significant increase in HSC numbers and enhanced their functions, such as having a higher engraftment capacity when transplanted into recipient mice compared to mice transplanted with PBS treated bone marrow cells. Furthermore, treating cells from the aorta-gonad-mesonephros (AGM)-site of HSC formation- of the E11.5 mouse embryo with rEGFL7 in vitro resulted in a higher percent of HSCs and primitive hematopoietic cells. EGFL7 is essential for embryogenic vascular formation, and its mRNA is expressed within the AGM3-5. Our group and others have also shown that EGFL7 is capable of binding to signaling receptors like Notch, which are important in normal hematopoiesis6,7. Specifically, Notch1 within the AGM has been shown to be critical during HSC formation from HSC precursors (HPs) (pro-HSC, pre-HSC type I and II) in the mouse embryo8,9. Here we hypothesize that the significant reduction of HSCs that we have observed in the adult Egfl7 -/- mice originate at the HPs, and the mechanism is at least in part being regulated by Notch1. We will test this hypothesis by isolating HPs from WT and Egfl7 -/- mice AGMs to assess their numbers and functions, as well as interrogating EGFL7-Notch1 interaction and downstream gene expression changes in the AGM.

Keywords:

98. Analysis of Adhesive Interactions in Isoforms of Cadherins Relevant for Lung Function and Hearing.

Daisy Alvarado (Biophysics ), Debadita Modak (Chemistry and Biochemistry )

Abstract:
The cadherin superfamily of calcium-dependent adhesion proteins has of over 100 members in the human genome. The biological function and biophysical properties of various members of the superfamily have been extensively studied in vivo and in vitro but understanding how alternative splicing alters their function remains unexplored in most cases. Here we have focused on two distinct non-clustered protocadherins that play a crucial role in cell-cell adhesion and sensory transduction and that have various isoforms resulting from gene splicing. The non-clustered Homo Sapiens (hs)protocadherin-1(PCDH1) is involved in respiratory function and it has an ectodomain composed of seven extracellular cadherin (EC) “repeats” that can mediate a trans (across cell membranes) adhesive bond formed by EC1-4 monomers arranged in an overlapped antiparallel complex. Intriguingly, hs PCDH1 has an isoform of unknown function that lacks the EC1-3 fragments. Likewise, the non-clustered Mus musculus (mm) cadherin-23 (CDH23) essential for hearing has an extracellular domain with 27 EC repeats and one of its isoforms lacks its adhesive repeats EC1-2 and most of the ectodomain. Here we have focused on testing the adhesive function of these two isoforms by producing C-terminal Fc tagged hsPCDH1 EC4-7 and mm CDH23 EC21-MAD28 protein fragments in HEK293 cells. Protein G beads were mixed with these fragments and used to investigate homophilic adhesion interactions, which were not observed. The biological function of these isoforms remains to be established.

Keywords: Protocadherins

99. Role of Unique C-terminal Domain in a Plant Aminoacyl-tRNA Trans-editing Protein

Jun-Kyu Byun (Ohio State Biochemistry Program), John Vu (Department of Chemistry and Biochemistry), Sio-Luan He (Department of Horticulture and Crop Science), Jyan-Chyun Jang (Department of Horticulture and Crop Science), Karin Musier-Forsyth (Department of Chemistry and Biochemistry)

Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing noncognate amino acids with cognate tRNAs. Many aaRSs have evolved quality control mechanisms to prevent mistranslation. Bacterial prolyl-tRNA synthetases (ProRSs) mischarge noncognate Ala onto tRNAPro and possess an insertion (INS) domain that can deacylate this mischarged tRNA and avoid mistranslation. Some bacteria and all eukaryotes lack an INS domain and instead, encode a free-standing trans-editing domain homolog, ProXp-ala. Sequence alignments revealed that all plant ProXp-ala contain a conserved C-terminal domain (CTD) of unknown structure and function that is missing from all other eukaryotic ProXp-ala. The CTD is predicted to fold into an all -helical domain. To determine the function of the CTD, we prepared a truncated Arabidopsis thaliana (At) ProXp-ala variant (ΔC-ProXp-ala). The in vitro Ala-tRNAPro deacylation rate of ΔC-ProXp-ala was decreased 16-fold relative to wild-type (WT) ProXp-ala. Electrophoretic mobility shift assays suggest that this decrease is primarily due to a tRNA binding defect. Size-exclusion chromatography revealed that WT At ProXp-ala adopts several oligomeric states, while ΔC-ProXp-ala is exclusively monomeric. The CTD may therefore function to enhance tRNA binding and/or induce multimerization in vitro. In vivo studies using split-YFP constructs support ProXp-ala homodimerization and show that the CTD is required for strong self-interaction. This work is expected to reveal the structure-function relationship of plant ProXp-ala in quality control of protein synthesis.

Keywords: aminoacyl-tRNA synthetase, trans-editing , plants

100. Title not available online - please see the booklet.

Christopher Gilbert (Ohio State Biochemistry Program, Biological Sciences Building 484 West 12th avenue, Columbus, OH 43210, USA), Jennifer Petrosino (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, 473 W 12th Ave, Columbus, OH, 43210, USA.), Federica Accornero (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, 473 W 12th Ave, Columbus, OH, 43210, USA.)

Abstract not available online - please check the booklet.

101. Sleep Fragmentation Following Traumatic Brain Injury Exacerbates Sub-Acute Behavioral Deficits and Transcriptional Changes at Chronic Time Points

Sam Houle (Department of Neuroscience, Institute for Behavioral Medicine Research), Zoe Tapp, Christopher Cotter, (Department of Neuroscience, Institute for Behavioral Medicine Research), Zach Zimomra (Institute for Behavioral Medicine Research), Siena Robertson, Yvanna Reyes, Sakeef Ahsan, Shannon Dobres, Jessica Mitsch (Department of Neuroscience, Institute for Behavioral Medicine Research), John Sheridan, Jonathan Godbout (Department of Neuroscience, Institute for Behavioral Medicine Research), Olga N. Kokiko-Cochran (Department of Neuroscience, Institute for Behavioral Medicine Research)

Abstract:
Stressful experiences elicit an immune response that is initially adaptive. However, persistent stress shifts the immune response to be detrimental. Stress-immune interactions may worsen long-term outcome in traumatic brain injury (TBI) survivors, who commonly have impaired stress response. Here, we leverage sleep fragmentation (SF) as a common physiological consequence of stress to study post-TBI neuroinflammation. We hypothesize that post-injury SF exacerbates TBI-induced sleep-wake disturbances, neuroinflammation, and cognitive impairments. Adult male and female mice received either a moderate lateral fluid percussion TBI or a sham injury. Mice were exposed to SF 5am-10am beginning one hour prior to light cycle or remained in control housing for 14 days post-injury (DPI). At 14 DPI mice were allowed to recover until 30 DPI. Sleep-wake activity was acquired for all mice using custom piezo-electric sensors for 30 DPI.

TBI increased sleep for 4 hours following injury. Unexpectedly, all mice exposed to SF slept more than control mice through 7 DPI. Post-TBI SF exacerbated spatial learning and memory deficits in the Morris water maze 14 DPI. By the last week of recovery SF mice slept comparably to control animals. However, transcriptional changes were present 30 DPI. Post-TBI SF increased transcription of cortical inflammation (Spi1, Abca1, Itga7), neurodegeneration (Abca1, Atg2b), and immunometabolic (Abca1, Lpl, Mef2a, Prkab2) genes. Additionally, transcripts of the gene Romo1 were increased by TBI SF indicating possible increased presence of reactive oxygen species 30 DPI. Canonical pathway analysis revealed that post-TBI SF upregulated the complement cascade, and nitric oxide and reactive oxygen species production in macrophages. Together, these results indicate that post-injury stress impairs the ability of the brain to adequately respond to and recover from injury.

Keywords: TBI, Neuroinflammation, Sleep

102. The Effect of High Fat Diet on Neuroinflammation, Synaptic Plasticity, and Pathology in the 3xTg-AD Mouse Model

Sabrina Mackey-Alfonso (NGP), Michael Butler (IBMR), Alberto Williams-Medina (IBMR), Nicholas Deems (NGP), Ashton Taylor (IBMR)

Abstract:
Alzheimer’s Disease (AD) is a neurodegenerative disease that presents with the hallmark pathology of neurofibrillary tau tangles and amyloid beta (AB) plaques and ultimately results in memory impairment. High fat diet (HFD) consumption has been shown to increase the risk of developing AD in humans and cause neuroinflammation and memory impairment in wild type animal models. However, the underlying mechanisms linking diet to AD risk is not well-studied. Thus, this project serves to investigate the effect of HFD consumption on 1) neuroinflammation and synaptic plasticity genes in the hippocampus and amygdala and 2) the severity of AD pathology in the hippocampus in a 3xTg-AD mouse model. Following the consumption of either standard chow or short-term HFD, mice (4-7 month-old) were transcardially saline-perfused and the hippocampus and amygdala were dissected for qPCR analysis of inflammatory and synaptic plasticity genes. In a separate cohort, mice were saline-perfused, and brains were post-fixed in 4% paraformaldehyde and processed for immunohistochemistry (DAB) staining of phosphorylated tau (AT8) and AB staining in the CA1 region of the hippocampus. Short-term HFD significantly increased the level of proinflammatory markers in the hippocampus and amygdala, including CD11b (p < 0.001), HMGB1 (p < 0.01), and TNF (p < 0.01). HFD also caused an alteration in synaptic plasticity genes in both the hippocampus and amygdala seen through an increase in a postsynaptic marker, PSD95, and a decrease in a presynaptic marker, synaptophysin (p<0.05). Our DAB data show HFD consumption increased the number and size of AT8 and AB puncta in the hippocampus of 3xTg-AD mice. These data suggest HFD consumption increases neuroinflammation, alters synaptic plasticity genes, and accelerates AD pathology in 3xTg-AD mice. Future studies will investigate the impact of HFD on learning and memory in AD mice and the cellular mechanisms driving these diet-induced inflammatory and pathological changes

Keywords: Alzheimers Disease, neuroinflammation

103. Probing the role of m6A RNA modification in the HIV-1 genomic RNA 5'UTR

Christina Ross (Department of Chemistry and Biochemistry and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210)

Abstract:
RNA modifications affect all aspects of RNA biology, likely impacting processes in viral replication such as RNA localization, stability, and assembly. To assemble virions, HIV-1 must select full-length genomic RNA (gRNA) from the cytoplasmic pool of cellular and viral RNA. Packaging is orchestrated by viral polyprotein Gag, which specifically interacts with the HIV-1 5'UTR RNA structure. Specific interactions between Gag and gRNA occur at sites in the packaging signal element (Psi) within the 5'UTR. Two N-6-methyladenosine (m6A) modifications have been previously identified in the 5'UTR, including one within Psi at an identified Gag binding site, and one in the tRNA primer binding site. We hypothesize m6A modifications in the HIV-1 5'UTR influence viral replication and assembly through shifting RNA structure and impacting tRNA and Gag interactions. We used splint ligation to construct Psi RNA, with the initial goal of producing specifically methylated Psi for binding studies. Salt-titration fluorescence anisotropy assays will be used to parse differences in specific and electrostatic Psi RNA-Gag interactions. In addition to binding, we aim to probe RNA structural differences between unmethylated and methylated RNA using native gel electrophoresis and RNA structure-probing. These studies may shed light on potential effects m6A has on retroviral packaging with implications for the development of therapeutics targeting this step in the HIV-1 lifecycle.

Keywords: HIV-1, m6A , RNA

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

Shridhar Sanghvi (Department of Molecular Cellular and Developmental Biology), Divya Sridharan (Department of Emergency Medicine), Vincenzo Trovato (Division of Cardiovascular Medicine, Department of Internal Medicine), Sakima Smith (Division of Cardiovascular Medicine, Department of Internal Medicine), Mahmood Khan (Department of Emergency Medicine), Harpreet Singh (Department of Physiology and Cell Biology)

Abstract not available online - please check the booklet.

105. DJ-1 prevents tau aggregation propensity through interaction with monomer

Daniela Jimenez-Harrison (Medical scientist training program, neuroscience graduate program ), Jeff Kuret (Department of Biological Chemistry and Pharmacology, Ohio State University )

Abstract not available online - please check the booklet.