Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells Elyse Nguyen, Depts. of Biology and Chemistry, Dipak Poria, & Esta Sterneck, with Dr. Sarah Williams, Dept. of Forensic Science Coordinated control of transcription and translation of gene expression impels cellular fate decision under different microenvironmental stresses. Cancer cells often usurp these regulatory machineries to adapt under microenvironmental stress or under therapeutic intervention. The transcription factor CEBPδ is induced by various stressors and mediates cellular adaptation and survival. RNA-seq analysis of a CEBPD-silenced human melanoma cell line, MB-435s, showed decreased expression of 12 aminoacyl-tRNA synthetase (aaRS) genes. Our group recently found that deletion of CEBPD by CRISPR/Cas9 (CEBPD-KO) compromised aminoacyl tRNA synthetase (aaRS) expression and global protein synthesis. However, despite this decrease in global protein production, the synthesis of certain proteins, such as ATF4, which promotes survival and/or death under stress conditions, is increased. Aminoacyl tRNA synthetases are essential enzymes in the process of protein synthesis which catalyze the addition of appropriate amino acid to its corresponding tRNA, and therefore act as a rate limiting step in cellular protein synthesis. In the current project, we sought to investigate the effect of silencing of specific aaRS genes, glutamyl-prolyl-tRNA synthetase (EPRS) and valyl-tRNA synthetase (VARS) on global protein translation and ATF4 expression. To address this question, we silenced the EPRS and VARS gene expression using two independent short-hairpin-RNA (shRNA) targeting two different regions of EPRS and VARS mRNAs in MB-435s cells. Silencing of EPRS gene showed compensatory upregulation of VARS and vice versa. Interestingly, our preliminary data suggested an upregulation of global protein synthesis after EPRS and VARS silencing in MB435s cells measured by puromycin pulse labelling. Ongoing experiments to validate the preliminary data and ATF4 expression will be discussed.https://scholarscompass.vcu.edu/uresposters/1326/thumbnail.jp

Medienkiller : Kodes eines medialen Mordes

Auf der Flucht wurde im Sommer 2001 das Ehepaar Manuela und Daniel Ruda von der Polizei gestellt. Beide bekannten sich zu dem gemeinschaftlich begangenen Mord an einem Bekannten im westfälischen Witten auf den sie genau 66-mal einstachen und einschlugen. Den Auftrag, so sagten sie übereinstimmend aus, habe ihnen Satan erteilt. Das Opfer sei so ”von seinem unwerten Leben befreit und von seiner Schmach erlöst worden”. Der Ablauf des Mordes, die darauf folgende Flucht und insbesondere auch die Gerichtsauftritte während des Prozesses waren voller Kodierungen, voller Bezüge auf Kult-Filme wie ’Natural Born Killers’ und Ikonen der Gothic-Szene. Eine Hommage an vermeintliche und reale Vorgänger in Zitaten und Symbolen, die weitgehend weder Justiz noch Presse entschlüsseln konnten. Bewusste Inszenenierungen in denen Manuela und Daniel Ruda ihre Rollen nicht nur spielten sondern lebten und dabei doch nur Kopien blieben

The Cebpd (C/EBPδ) Gene Is Induced by Luteinizing Hormones in Ovarian Theca and Interstitial Cells But Is Not Essential for Mouse Ovary Function

The CCAAT/enhancer binding protein (CEBP) family of transcription factors includes five genes. In the ovary, both Cebpa and Cebpb are essential for granulosa cell function. In this study we have explored the role of the Cebpd gene in ovarian physiology by expression and functional studies. Here we report that Cebpd (C/EBPδ) is expressed in the mouse ovary in a highly restricted temporal and spatial pattern. In response to luteinizing hormone (LH/hCG), CEBPD expression is transiently induced in interstitial cells and in theca cells of follicles from the primary to pre-ovulatory stage, and overlaps in part with expression of the alpha-smooth muscle actin protein. Efficient down-regulation of CEBPD was dependent on a functional Cebpb gene. Proliferating human theca cells in culture also express Cebpd. Cells from patients with polycystic ovarian syndrome (PCOS) exhibited higher Cebpd expression levels. However, deletion of Cebpd in mice had no overt effect on ovarian physiology and reproductive function. Very little is known at present about the molecular mechanisms underlying theca/interstitial cell functions. The expression pattern of CEBPD reported here identifies a novel functional unit of mouse theca cells of primary through tertiary follicles responding to LH/hCG together with a subset of interstitial cells. This acute stimulation of CEBPD expression may be exploited to further characterize the hormonal regulation and function of theca and interstitial cells

Modeling, Calibration, and Evaluation of a Tendon-Actuated Planar Parallel Continuum Robot

In this work, a novel planar parallel continuum robot (PCR) is introduced, consisting of three kinematic chains that are coupled at a triangular end-effector platform and include tendon-actuated continuum segments. The kinematics of the resulting structure are derived by adapting the descriptions for conventional planar parallel manipulators to include constant curvature bending of the utilized continuous segments. To account for friction and non-linear material effects, a data-driven model is used to relate tendon displacements and curvature of the utilized continuum segments. A calibration of the derived kinematic model is conducted to specifically represent the constructed prototype. This includes the calibration of geometric parameters for each kinematic chain and for the end-effector platform. During evaluation, positioning repeatability of 1.0% in relation to one continuum segment length of the robot, and positioning accuracy of 1.4%, are achieved. These results are comparable to commonly used kineto-static modeling approaches for PCR. The presented model achieves high path accuracies regarding the robot's end-effector pose in an open-loop control scenario

Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB

Disequilibrium between bone-forming osteoblasts and bone-resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancer-binding protein beta (C/EBPbeta) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP(*), LAP) and a short repressive (LIP) isoforms from a single C/EBPbeta transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPbeta null (c/ebpbeta(-/-)) or in LIP knock-in (L/L) osteoclast precursors. C/EBPbeta mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPbeta isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression