Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

PubMed

Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

2016-08-26

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fred, Rikard G., E-mail: Rikard.Fred@mcb.uu.se; Sandberg, Monica; Pelletier, Jerry

Highlights: {yields} The polypyrimidine tract binding protein binds to the 5'-UTR of the insulin mRNA. {yields} Insulin mRNA can be translated via a cap-independent mechanism. {yields} The fraction cap-independent insulin synthesis increases during conditions of stress. {yields} The {beta}-cell is able to uphold basal insulin biosynthesis under conditions of stress. -- Abstract: The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRESmore » trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

PubMed

Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

2010-04-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

PubMed Central

Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

2010-01-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

A Dual Interaction Between the 5'- and 3'-Ends of the Melon Necrotic Spot Virus (MNSV) RNA Genome Is Required for Efficient Cap-Independent Translation.

PubMed

Miras, Manuel; Rodríguez-Hernández, Ana M; Romero-López, Cristina; Berzal-Herranz, Alfredo; Colchero, Jaime; Aranda, Miguel A; Truniger, Verónica

2018-01-01

In eukaryotes, the formation of a 5'-cap and 3'-poly(A) dependent protein-protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3'-CITE (for cap-independent translation enhancer), a RNA element present in their 3'-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3'-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5'- and 3'-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3'-CITE to the 5'-end. For the carmovirus melon necrotic spot virus (MNSV), a 3'-CITE has been identified, and the presence of its 5'-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5'-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3'-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3'-CITE and at least one complementary sequence in the 5'-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5'-3' RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.

Human Merkel cell polyomavirus small T antigen is an oncoprotein targeting the 4E-BP1 translation regulator

PubMed Central

Shuda, Masahiro; Kwun, Hyun Jin; Feng, Huichen; Chang, Yuan; Moore, Patrick S.

2011-01-01

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorage- and contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT–associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers. PMID:21841310

3′ Cap-Independent Translation Enhancers of Plant Viruses

PubMed Central

Simon, Anne E.; Miller, W. Allen

2014-01-01

In the absence of a 5′ cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3′ untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3′ cap-independent translational enhancers (3′ CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3′ CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5′ end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3′ CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus’s host range. PMID:23682606

Structural and Functional Diversity of Plant Virus 3'-Cap-Independent Translation Enhancers (3'-CITEs).

PubMed

Truniger, Verónica; Miras, Manuel; Aranda, Miguel A

2017-01-01

Most of the positive-strand RNA plant viruses lack the 5'-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5'- or 3'-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3'-end of viruses belonging to the family Tombusviridae and the genus Luteovirus . Seven classes of 3'-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3'-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5'-end by different mechanisms, often long-distance RNA-RNA interactions. As previously proposed and recently found in one case in nature, 3'-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3'-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3'-CITEs belonging to different classes.

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

PubMed

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

PubMed Central

Meng, Zheng; King, Peter H.; Nabors, L. Burt; Jackson, Nateka L.; Chen, Ching-Yi; Emanuel, Peter D.; Blume, Scott W.

2005-01-01

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: evaluation of the Trojan horse model for TYMV RNA translation.

PubMed

Matsuda, Daiki; Dreher, Theo W

2007-01-01

Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3'-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3' TLS but is stimulated by the presence of a 5'-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model.

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: Evaluation of the Trojan horse model for TYMV RNA translation

PubMed Central

Matsuda, Daiki; Dreher, Theo W.

2007-01-01

Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3′-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3′ TLS but is stimulated by the presence of a 5′-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNAi Met involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model. PMID:17095542

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

PubMed

Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

2015-06-01

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped RNA

PubMed Central

Lacerda, Livia; Benda, Christian; Holze, Cathleen; Eberl, Christian H.; Mann, Angelika; Kindler, Eveline; Gil-Cruz, Cristina; Ziebuhr, John; Thiel, Volker; Pichlmair, Andreas

2013-01-01

Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells. PMID:24098121

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

PubMed Central

Kowalska, Joanna; Wypijewska del Nogal, Anna; Darzynkiewicz, Zbigniew M.; Buck, Janina; Nicola, Corina; Kuhn, Andreas N.; Lukaszewicz, Maciej; Zuberek, Joanna; Strenkowska, Malwina; Ziemniak, Marcin; Maciejczyk, Maciej; Bojarska, Elzbieta; Rhoads, Robert E.; Darzynkiewicz, Edward; Sahin, Ugur; Jemielity, Jacek

2014-01-01

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization. PMID:25150148

Structural and Functional Diversity of Plant Virus 3′-Cap-Independent Translation Enhancers (3′-CITEs)

PubMed Central

Truniger, Verónica; Miras, Manuel; Aranda, Miguel A.

2017-01-01

Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes. PMID:29238357

La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

PubMed Central

Philippe, Lucas; Vasseur, Jean-Jacques; Debart, Françoise

2018-01-01

Abstract Cell growth is a complex process shaped by extensive and coordinated changes in gene expression. Among these is the tightly regulated translation of a family of growth-related mRNAs defined by a 5′ terminal oligopyrimidine (TOP) motif. TOP mRNA translation is partly controlled via the eukaryotic initiation factor 4F (eIF4F), a translation factor that recognizes the mRNA 5′ cap structure. Recent studies have also implicated La-related protein 1 (LARP1), which competes with eIF4F for binding to mRNA 5′ ends. However, it has remained controversial whether LARP1 represses TOP mRNA translation directly and, if so, what features define its mRNA targets. Here, we show that the C-terminal half of LARP1 is necessary and sufficient to control TOP mRNA translation in cells. This fragment contains the DM15 cap-binding domain as well as an adjacent regulatory region that we identified. We further demonstrate that purified LARP1 represses TOP mRNA translation in vitro through the combined recognition of both the TOP sequence and cap structure, and that its intrinsic repressive activity and affinity for these features are subject to regulation. These results support a model whereby the translation of TOP mRNAs is controlled by a growth-regulated competition between eIF4F and LARP1 for their 5′ ends. PMID:29244122

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation

PubMed Central

Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A.; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J.

2015-01-01

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host:pathogen interface. In this study we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine (m7G) mRNA cap, increased the total level of protein synthesis, and co-localized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a non-viral reporter gene and increased the translation of a reporter containing an HCMV 5’ untranslated region (5’UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5’UTR required the pTRS1 RNA and PKR binding domains, and was likely the result of PKR inhibition. However pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5’UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. PMID:25894605

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

PubMed

Carnicelli, Domenica; Arfilli, Valentina; Onofrillo, Carmine; Alfieri, Roberta R; Petronini, Pier Giorgio; Montanaro, Lorenzo; Brigotti, Maurizio

2017-02-12

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5'-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Cap-independent translation of human SP-A 5′-UTR variants: a double-loop structure and cis-element contribution

PubMed Central

Wang, Guirong; Guo, Xiaoxuan; Silveyra, Patricia; Kimball, Scot R.; Floros, Joanna

2009-01-01

Human surfactant protein A (hSP-A), a molecule of innate immunity and surfactant-related functions, consists of two functional genes, SP-A1 and SP-A2. SP-A expression is regulated by several factors including environmental stressors. SP-A1 and SP-A2 5′-untranslated region (5′-UTR) splice variants have a differential impact on translation efficiency and mRNA stability. To study whether these variants mediate internal ribosome entry site (IRES) activity (i.e., cap-independent translation), we performed transient transfection experiments in H441 cells with constructs containing one SP-A1 (A′D′, AB′D′, or A′CD′) or SP-A2 (ABD) 5′-UTR splice variant between the Renilla and firefly luciferase genes of a bicistronic reporter vector. We found that 1) variants A′D′, ABD, and AB′D′ exhibit significantly higher IRES activities than negative control (no SP-A 5′-UTR) and A′CD′ has no activity; the order of highest IRES activity was ABD > A′D′ > AB′D; 2) IRES activity of ABD significantly increased in response to diesel particulate matter (20 μg/ml) but not in response to ozone (1 ppm for 1 h); 3) deletion mutants of ABD revealed regulatory elements associated with IRES activity; one at the end of exon A attenuated activity, whereas a region containing a short adenosine-rich motif in the second half of exon B and the start of exon D enhanced activity; 4) elimination of a predicted double-loop structure or increase in free energy significantly reduced IRES activity; 5) elimination of one or both double-loop structures in A′D′ did not affect cap-dependent translation activity. Thus several factors, including cis-elements and secondary structure type and stability, are required for hSP-A 5′-UTR variant-mediated cap-independent translation. PMID:19181744

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

PubMed

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Protein Translation and Signaling in Human Eosinophils

PubMed Central

Esnault, Stephane; Shen, Zhong-Jian; Malter, James S.

2017-01-01

We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases. PMID:28971096

Rapid, experience-dependent translation of neurogranin enables memory encoding.

PubMed

Jones, Kendrick J; Templet, Sebastian; Zemoura, Khaled; Kuzniewska, Bozena; Pena, Franciso X; Hwang, Hongik; Lei, Ding J; Haensgen, Henny; Nguyen, Shannon; Saenz, Christopher; Lewis, Michael; Dziembowska, Magdalena; Xu, Weifeng

2018-06-19

Experience induces de novo protein synthesis in the brain and protein synthesis is required for long-term memory. It is important to define the critical temporal window of protein synthesis and identify newly synthesized proteins required for memory formation. Using a behavioral paradigm that temporally separates the contextual exposure from the association with fear, we found that protein synthesis during the transient window of context exposure is required for contextual memory formation. Among an array of putative activity-dependent translational neuronal targets tested, we identified one candidate, a schizophrenia-associated candidate mRNA, neurogranin (Ng, encoded by the Nrgn gene) responding to novel-context exposure. The Ng mRNA was recruited to the actively translating mRNA pool upon novel-context exposure, and its protein levels were rapidly increased in the hippocampus. By specifically blocking activity-dependent translation of Ng using virus-mediated molecular perturbation, we show that experience-dependent translation of Ng in the hippocampus is required for contextual memory formation. We further interrogated the molecular mechanism underlying the experience-dependent translation of Ng, and found that fragile-X mental retardation protein (FMRP) interacts with the 3'UTR of the Nrgn mRNA and is required for activity-dependent translation of Ng in the synaptic compartment and contextual memory formation. Our results reveal that FMRP-mediated, experience-dependent, rapid enhancement of Ng translation in the hippocampus during the memory acquisition enables durable context memory encoding. Copyright © 2018 the Author(s). Published by PNAS.

Rapid, experience-dependent translation of neurogranin enables memory encoding

PubMed Central

Jones, Kendrick J.; Templet, Sebastian; Zemoura, Khaled; Pena, Franciso X.; Hwang, Hongik; Lei, Ding J.; Haensgen, Henny; Nguyen, Shannon; Saenz, Christopher; Lewis, Michael; Dziembowska, Magdalena

2018-01-01

Experience induces de novo protein synthesis in the brain and protein synthesis is required for long-term memory. It is important to define the critical temporal window of protein synthesis and identify newly synthesized proteins required for memory formation. Using a behavioral paradigm that temporally separates the contextual exposure from the association with fear, we found that protein synthesis during the transient window of context exposure is required for contextual memory formation. Among an array of putative activity-dependent translational neuronal targets tested, we identified one candidate, a schizophrenia-associated candidate mRNA, neurogranin (Ng, encoded by the Nrgn gene) responding to novel-context exposure. The Ng mRNA was recruited to the actively translating mRNA pool upon novel-context exposure, and its protein levels were rapidly increased in the hippocampus. By specifically blocking activity-dependent translation of Ng using virus-mediated molecular perturbation, we show that experience-dependent translation of Ng in the hippocampus is required for contextual memory formation. We further interrogated the molecular mechanism underlying the experience-dependent translation of Ng, and found that fragile-X mental retardation protein (FMRP) interacts with the 3′UTR of the Nrgn mRNA and is required for activity-dependent translation of Ng in the synaptic compartment and contextual memory formation. Our results reveal that FMRP-mediated, experience-dependent, rapid enhancement of Ng translation in the hippocampus during the memory acquisition enables durable context memory encoding. PMID:29880715

Dendritic mRNA targeting and translation.

PubMed

Kindler, Stefan; Kreienkamp, Hans-Jürgen

2012-01-01

Selective targeting of specific mRNAs into neuronal dendrites and their locally regulated translation at particular cell contact sites contribute to input-specific synaptic plasticity. Thus, individual synapses become decision-making units, which control gene expression in a spatially restricted and nucleus-independent manner. Dendritic targeting of mRNAs is achieved by active, microtubule-dependent transport. For this purpose, mRNAs are packaged into large ribonucleoprotein (RNP) particles containing an array of trans-acting RNA-binding proteins. These are attached to molecular motors, which move their RNP cargo into dendrites. A variety of proteins may be synthesized in dendrites, including signalling and scaffold proteins of the synapse and neurotransmitter receptors. In some cases, such as the alpha subunit of the calcium/calmodulin-dependent protein kinase II (αCaMKII) and the activity-regulated gene of 3.1 kb (Arg3.1, also referred to as activity-regulated cDNA, Arc), their local synthesis at synapses can modulate long-term changes in synaptic efficiency. Local dendritic translation is regulated by several signalling cascades including Akt/mTOR and Erk/MAP kinase pathways, which are triggered by synaptic activity. More recent findings show that miRNAs also play an important role in protein synthesis at synapses. Disruption of local translation control at synapses, as observed in the fragile X syndrome (FXS) and its mouse models and possibly also in autism spectrum disorders, interferes with cognitive abilities in mice and men.

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism.

PubMed

Kunze, Michael M; Benz, Fabienne; Brauß, Thilo F; Lampe, Sebastian; Weigand, Julia E; Braun, Johannes; Richter, Florian M; Wittig, Ilka; Brüne, Bernhard; Schmid, Tobias

2016-07-01

Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5'UTR of sST2. Furthermore, we found that MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation. Determination of the sST2-5'UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5'UTR to be required for optimal IRES activity. Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5'UTR, acts as an ITAF, and thus controls the activity of the sST2-IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5'UTR contains an IRES element, which is activated by a MEK/ERK-dependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation

PubMed Central

Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth A.; Contreras, Nataly; Hertz, Marla I.; Olivares, Eduardo; Cáceres, C. Joaquín; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R.; López-Lastra, Marcelo

2016-01-01

The 5′leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work we examine the internal ribosome entry site (IRES) located in the 5′leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5′leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

PubMed Central

Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

2014-01-01

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene

Glucocorticoids inhibit coordinated translation of. cap alpha. - and. beta. -globin mRNAs in Friend erythroleukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papaconstantinou, J.; Stewart, J.A.; Rabek, J.P.

The dimethylsulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend erythroleukemia cells is inhibited by the glucocorticoids hydrocortisone, dexamethasone, and fluocinolone acetonide; hydrocortisone, at concentrations of 10/sup -5/ to 10/sup -8/ M inhibits by 90-30% and fluocinolone acetonide at concentrations of 10/sup -8/ to 10/sup -11/ M shows a greater than 90% inhibition. At these concentrations the hormones have no effect on cell growth or viability. In this study it has been shown that there is a group of proteins, including the ..cap alpha..- and ..beta..-globins, whose regulation is associated with the induction of Friend erythroleukemia cell differentiation, and thatmore » the expression of these, in addition to ..cap alpha..- and ..beta..-globin, is affected by glucocorticoids. It is concluded that, although the translation of ..cap alpha..- and ..beta..-globin mRNA is a major site of inhibition by glucocorticoids, there is a detectable amount of ..cap alpha..- and ..beta..-globin mRNA translation which results in unequal amounts of globin synthesis and an overall more potent inhibition of hemoglobin formation.« less

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

PubMed

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

PubMed Central

Strenkowska, Malwina; Grzela, Renata; Majewski, Maciej; Wnek, Katarzyna; Kowalska, Joanna; Lukaszewicz, Maciej; Zuberek, Joanna; Darzynkiewicz, Edward; Kuhn, Andreas N.; Sahin, Ugur; Jemielity, Jacek

2016-01-01

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5′ end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization. PMID:27903882

Translational Approaches Targeting Reconsolidation

PubMed Central

Kroes, Marijn C.W.; LeDoux, Joseph E.; Phelps, Elizabeth A.

2017-01-01

Maladaptive learned responses and memories contribute to psychiatric disorders that constitute a significant socio-economic burden. Primary treatment methods teach patients to inhibit maladaptive responses, but do not get rid of the memory itself, which explains why many patients experience a return of symptoms even after initially successful treatment. This highlights the need to discover more persistent and robust techniques to diminish maladaptive learned behaviours. One potentially promising approach is to alter the original memory, as opposed to inhibiting it, by targeting memory reconsolidation. Recent research shows that reactivating an old memory results in a period of memory flexibility and requires restorage, or reconsolidation, for the memory to persist. This reconsolidation period allows a window for modification of a specific old memory. Renewal of memory flexibility following reactivation holds great clinical potential as it enables targeting reconsolidation and changing of specific learned responses and memories that contribute to maladaptive mental states and behaviours. Here, we will review translational research on non-human animals, healthy human subjects, and clinical populations aimed at altering memories by targeting reconsolidation using biological treatments (electrical stimulation, noradrenergic antagonists) or behavioural interference (reactivation–extinction paradigm). Both approaches have been used successfully to modify aversive and appetitive memories, yet effectiveness in treating clinical populations has been limited. We will discuss that memory flexibility depends on the type of memory tested and the brain regions that underlie specific types of memory. Further, when and how we can most effectively reactivate a memory and induce flexibility is largely unclear. Finally, the development of drugs that can target reconsolidation and are safe for use in humans would optimize cross-species translations. Increasing the understanding of

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

PubMed

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

PubMed

Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

2016-07-01

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. © 2016 Federation of European Biochemical Societies.

Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration.

PubMed

Lin, Tiffany V; Hsieh, Lawrence; Kimura, Tomoki; Malone, Taylor J; Bordey, Angélique

2016-10-04

Hyperactive mammalian target of rapamycin complex 1 (mTORC1) is a shared molecular hallmark in several neurodevelopmental disorders characterized by abnormal brain cytoarchitecture. The mechanisms downstream of mTORC1 that are responsible for these defects remain unclear. We show that focally increasing mTORC1 activity during late corticogenesis leads to ectopic placement of upper-layer cortical neurons that does not require altered signaling in radial glia and is accompanied by changes in layer-specific molecular identity. Importantly, we found that decreasing cap-dependent translation by expressing a constitutively active mutant of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) prevents neuronal misplacement and soma enlargement, while partially rescuing dendritic hypertrophy induced by hyperactive mTORC1. Furthermore, overactivation of translation alone through knockdown of 4E-BP2 was sufficient to induce neuronal misplacement. These data show that many aspects of abnormal brain cytoarchitecture can be prevented by manipulating a single intracellular process downstream of mTORC1, cap-dependent translation.

Emerging Therapeutics Targeting mRNA Translation

PubMed Central

Malina, Abba; Mills, John R.; Pelletier, Jerry

2012-01-01

A defining feature of many cancers is deregulated translational control. Typically, this occurs at the level of recruitment of the 40S ribosomes to the 5′-cap of cellular messenger RNAs (mRNAs), the rate-limiting step of protein synthesis, which is controlled by the heterotrimeric eukaryotic initiation complex eIF4F. Thus, eIF4F in particular, and translation initiation in general, represent an exploitable vulnerability and unique opportunity for therapeutic intervention in many transformed cells. In this article, we discuss the development, mode of action and biological activity of a number of small-molecule inhibitors that interrupt PI3K/mTOR signaling control of eIF4F assembly, as well as compounds that more directly block eIF4F activity. PMID:22474009

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

PubMed

Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

2016-02-12

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Translational control of aberrant stress responses as a hallmark of cancer.

PubMed

El-Naggar, Amal M; Sorensen, Poul H

2018-04-01

Altered mRNA translational control is emerging as a critical factor in cancer development and progression. Targeting specific elements of the translational machinery, such as mTORC1 or eIF4E, is emerging as a new strategy for innovative cancer therapy. While translation of most mRNAs takes place through cap-dependent mechanisms, a sub-population of cellular mRNA species, particularly stress-inducible mRNAs with highly structured 5'-UTR regions, are primarily translated through cap-independent mechanisms. Intriguingly, many of these mRNAs encode proteins that are involved in tumour cell adaptation to microenvironmental stress, and thus linked to aggressive behaviour including tumour invasion and metastasis. This necessitates a rigorous search for links between microenvironmental stress and aggressive tumour phenotypes. Under stress, cells block global protein synthesis to preserve energy while maintaining selective synthesis of proteins that support cell survival. One highly conserved mechanism to regulate protein synthesis under cell stress is to sequester mRNAs into cytosolic aggregates called stress granules (SGs), where their translation is silenced. SGs confer survival advantages and chemotherapeutic resistance to tumour cells under stress. Recently, it has been shown that genetically blocking SG formation dramatically reduces tumour invasive and metastatic capacity in vivo. Therefore, targeting SG formation might represent a potential treatment strategy to block cancer metastasis. Here, we present the critical link between selective mRNA translation, stress adaptation, SGs, and tumour progression. Further, we also explain how deciphering mechanisms of selective mRNA translation occurs under cell stress holds great promise for the identification of new targets in the treatment of cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland

Interplanetary magnetic field dependency of stable Sun-aligned polar cap arcs

NASA Technical Reports Server (NTRS)

Valladares, C. E.; Carlson, H. C., Jr.; Fukui, K.

1994-01-01

This is the first analysis, using a statistically significant data set, of the morphological dependence of the presence, orientation, and motion of stable sun-aligned polar cap arcs upon the vector interplanetary magnetic field (IMF). For the one winter season analyzed we had 1392 all-sky 630.0-nm images of 2-min resolution containing a total of 150 polar cap arcs, all with corresponding values of the IMF as measured by International Monitoring Platform (IMP) 8 or International Sun Earth Explorer (ISEE) 2. After demonstrating an unbiased data set with smooth normal distributions of events versus the dimensions of time, space, and IMF component, we examine IMF dependencies of the properties of the optical arcs. A well-defined dependence for B(sub z) is found for the presence/absence of stable Sun-aligned polar cap arcs. Consistent with previous statistical studies, the probability of observing polar cap aurora steadily increases for larger positive values of B(sub z), and linearly decreases when B(sub z) becomes more negative. The probability of observing Sun-aligned arcs within the polar cap is determined to vary sharply as a function of the arc location; arcs were observed 40% of the time on the dawnside and only 10% on the duskside. This implies an overall probability of at least 40% for the whole polar cap. 20% of the arcs were observed during 'southward IMF conditions,' but in fact under closer inspection were found to have been formed under northward IMF conditions; these 'residual' positive B(sub z) arcs ha d a delayed residence time in the polar cap of about what would be expected after a north to south transition of B(sub z). A firm dependence on B(sub y) is also found for both the orientation and the dawn-dusk direction of motion of the arcs. All the arcs are Sun-aligned to a first approximation, but present deviations from this orientation, depending primarily upon the location of the arc in corrected geomagnetic (CG) coordinates. The arcs populating the

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

PubMed

Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

2007-01-01

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

PubMed Central

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

PubMed

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

RACK1-mediated translation control promotes liver fibrogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Min; Peng, Peike; Wang, Jiajun

Activation of quiescent hepatic stellate cells (HSCs) is the central event of liver fibrosis. The translational machinery is an optimized molecular network that affects cellular homoeostasis and diseases, whereas the role of protein translation in HSCs activation and liver fibrosis is little defined. Our previous report suggests that up-regulation of receptor for activated C-kinase 1(RACK1) in HSCs is critical for liver fibrogenesis. In this study, we found that RACK1 promoted macrophage conditioned medium (MCM)-induced assembly of eIF4F and phosphorylation of eIF4E in primary HSCs. RACK1 enhanced the translation and expression of pro-fibrogenic factors collagen 1α1, snail and cyclin E1 inducedmore » by MCM. Administration of PP242 or knock-down of eIF4E suppressed RACK1-stimulated collagen 1α1 production, proliferation and migration in primary HSCs. In addition, depletion of eIF4E attenuated thioacetamide (TAA)-induced liver fibrosis in vivo. Our data suggest that RACK1-mediated stimulation of cap-dependent translation plays crucial roles in HSCs activation and liver fibrogenesis, and targeting translation initiation could be a promising strategy for the treatment of liver fibrosis. - Highlights: • RACK1 induces the assembly of eIF4F and phosphorylation of eIF4E in primary HSCs. • RACK1 stimulates the translation of collagen 1α1, snail and cyclin E1 in HSCs. • RACK1 promotes HSCs activation via cap-mediated translation. • Depletion of eIF4E suppresses liver fibrogenesis in vivo.« less

Effects of audio-visual presentation of target words in word translation training

NASA Astrophysics Data System (ADS)

Akahane-Yamada, Reiko; Komaki, Ryo; Kubo, Rieko

2004-05-01

Komaki and Akahane-Yamada (Proc. ICA2004) used 2AFC translation task in vocabulary training, in which the target word is presented visually in orthographic form of one language, and the appropriate meaning in another language has to be chosen between two choices. Present paper examined the effect of audio-visual presentation of target word when native speakers of Japanese learn to translate English words into Japanese. Pairs of English words contrasted in several phonemic distinctions (e.g., /r/-/l/, /b/-/v/, etc.) were used as word materials, and presented in three conditions; visual-only (V), audio-only (A), and audio-visual (AV) presentations. Identification accuracy of those words produced by two talkers was also assessed. During pretest, the accuracy for A stimuli was lowest, implying that insufficient translation ability and listening ability interact with each other when aurally presented word has to be translated. However, there was no difference in accuracy between V and AV stimuli, suggesting that participants translate the words depending on visual information only. The effect of translation training using AV stimuli did not transfer to identification ability, showing that additional audio information during translation does not help improve speech perception. Further examination is necessary to determine the effective L2 training method. [Work supported by TAO, Japan.

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

PubMed

Tehrani, A M; Hwang, S-K; Kim, T-H; Cho, C-S; Hua, J; Nah, W-S; Kwon, J-T; Kim, J-S; Chang, S-H; Yu, K-N; Park, S-J; Bhandari, D R; Lee, K-H; An, G-H; Beck, G R; Cho, M-H

2007-03-01

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

PubMed

Hou, Jiun-Nan; Chen, Tien-Huang; Chiang, Yi-Hsuan; Peng, Jing-Yun; Yang, Tsong-Han; Cheng, Chih-Chieh; Sofiyatun, Eny; Chiu, Cheng-Hsun; Chiang-Ni, Chuan; Chen, Wei-June

2017-09-20

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication

PubMed Central

Hou, Jiun-Nan; Chen, Tien-Huang; Chiang, Yi-Hsuan; Peng, Jing-Yun; Yang, Tsong-Han; Cheng, Chih-Chieh; Sofiyatun, Eny; Chiu, Cheng-Hsun; Chiang-Ni, Chuan; Chen, Wei-June

2017-01-01

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5′-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5′-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK

Loss of PINK1 attenuates HIF-1α induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.

PubMed

Lin, William; Wadlington, Natasha L; Chen, Linan; Zhuang, Xiaoxi; Brorson, James R; Kang, Un Jung

2014-02-19

Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1(-/-) mouse cells exhibited significantly reduced induction of HIF-1α protein, HIF-1α transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1α pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

PubMed Central

Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

2016-01-01

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development.

PubMed

Schratt, Gerhard M; Nigh, Elizabeth A; Chen, Wen G; Hu, Linda; Greenberg, Michael E

2004-08-18

Local regulation of mRNA translation plays an important role in axon guidance, synaptic development, and neuronal plasticity. Little is known, however, regarding the mechanisms that control translation in neurons, and only a few mRNAs have been identified that are locally translated within axon and dendrites. Using Affymetrix gene arrays to identify mRNAs that are newly associated with polysomes after exposure to BDNF, we identified subsets of mRNAs for which translation is enhanced in neurons at different developmental stages. In mature neurons, many of these mRNAs encode proteins that are known to function at synapses, including CamKIIalpha, NMDA receptor subunits, and the postsynaptic density (PSD) scaffolding protein Homer2. BDNF regulates the translation of Homer2 locally in the synaptodendritic compartment by activating translational initiation via a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway. These findings suggest that BDNF likely regulates synaptic function by inducing the local synthesis of numerous synaptic proteins. The local translation of the cytoskeleton-associated protein Homer2 in particular might have important implications for growth cone dynamics and dendritic spine development.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

PubMed

Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

2017-09-19

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A New Framework for Understanding IRES-mediated Translation

PubMed Central

Komar, Anton A.; Mazumder, Barsanjit; Merrick, William C.

2012-01-01

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m7G-assisted internal initiation). PMID:22555019

Translational Regulation of PTEN/MMAC1 Expression in Prostate Cancer

DTIC Science & Technology

2003-05-01

the transcription of dicistronic RNA encoding Renilla luciferase as the first cistron and firefly luciferase as the second cistron. Translation of the...first cistron ( Renilla luciferase) serves as an indicator of cap-dependent translation while translation of the second cistron (firefly luciferase...cellular IRES (Sachs, 2000). These dicistronic constructs were transfected into HeLa cells and both Renilla and firefly luciferase activities were measured

Hypermethylated-capped selenoprotein mRNAs in mammals

PubMed Central

Wurth, Laurence; Gribling-Burrer, Anne-Sophie; Verheggen, Céline; Leichter, Michael; Takeuchi, Akiko; Baudrey, Stéphanie; Martin, Franck; Krol, Alain; Bertrand, Edouard; Allmang, Christine

2014-01-01

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo. PMID:25013170

Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome

PubMed Central

Bolinger, Cheryl; Boris-Lawrie, Kathleen

2009-01-01

Retroviruses have evolved multiple strategies to direct the synthesis of a complex proteome from a single primary transcript. Their mechanisms are modulated by a breadth of virus-host interactions, which are of significant fundamental interest because they ultimately affect the efficiency of virus replication and disease pathogenesis. Motifs located within the untranslated region (UTR) of the retroviral RNA have established roles in transcriptional trans-activation, RNA packaging, and genome reverse transcription; and a growing literature has revealed a necessary role of the UTR in modulating the efficiency of viral protein synthesis. Examples include a 5' UTR post-transcriptional control element (PCE), present in at least eight retroviruses, that interacts with cellular RNA helicase A to facilitate cap-dependent polyribosome association; and 3' UTR constitutive transport element (CTE) of Mason-Pfizer monkey virus that interacts with Tap/NXF1 and SR protein 9G8 to facilitate RNA export and translational utilization. By contrast, nuclear protein hnRNP E1 negatively modulates HIV-1 Gag, Env, and Rev protein synthesis. Alternative initiation strategies by ribosomal frameshifting and leaky scanning enable polycistronic translation of the cap-dependent viral transcript. Other studies posit cap-independent translation initiation by internal ribosome entry at structural features of the 5' UTR of selected retroviruses. The retroviral armamentarium also commands mechanisms to counter cellular post-transcriptional innate defenses, including protein kinase R, 2',5'-oligoadenylate synthetase and the small RNA pathway. This review will discuss recent and historically-recognized insights into retrovirus translational control. The expanding knowledge of retroviral post-transcriptional control is vital to understanding the biology of the retroviral proteome. In a broad perspective, each new insight offers a prospective target for antiviral therapy and strategic improvement of gene

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2.

PubMed

Wang, Fengping; Qiu, Ye; Zhang, Huifang M; Hanson, Paul; Ye, Xin; Zhao, Guangze; Xie, Ronald; Tong, Lei; Yang, Decheng

2017-07-01

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70-overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans-acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap-dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt-mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K- and cell division cycle protein 2 homolog (Cdc2)-mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection. © 2017 John Wiley & Sons Ltd.

NAT1/DAP5/p97 and Atypical Translational Control in the Drosophila Circadian Oscillator

PubMed Central

Bradley, Sean; Narayanan, Siddhartha; Rosbash, Michael

2012-01-01

Circadian rhythms are driven by gene expression feedback loops in metazoans. Based on the success of genetic screens for circadian mutants in Drosophila melanogaster, we undertook a targeted RNAi screen to study the impact of translation control genes on circadian locomotor activity rhythms in flies. Knockdown of vital translation factors in timeless protein-positive circadian neurons caused a range of effects including lethality. Knockdown of the atypical translation factor NAT1 had the strongest effect and lengthened circadian period. It also dramatically reduced PER protein levels in pigment dispersing factor (PDF) neurons. BELLE (BEL) protein was also reduced by the NAT1 knockdown, presumably reflecting a role of NAT1 in belle mRNA translation. belle and NAT1 are also targets of the key circadian transcription factor Clock (CLK). Further evidence for a role of NAT1 is that inhibition of the target of rapamycin (TOR) kinase increased oscillator activity in cultured wings, which is absent under conditions of NAT1 knockdown. Moreover, the per 5′- and 3′-UTRs may function together to facilitate cap-independent translation under conditions of TOR inhibition. We suggest that NAT1 and cap-independent translation are important for per mRNA translation, which is also important for the circadian oscillator. A circadian translation program may be especially important in fly pacemaker cells. PMID:22904033

Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

PubMed

Kleene, Kenneth C

2016-03-01

Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

PubMed Central

Sato, Hanae; Maquat, Lynne E.

2009-01-01

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured. PMID:19884259

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

PubMed Central

Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

2016-01-01

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

NASA Astrophysics Data System (ADS)

Lindemann, Stephan W.; Yost, Christian C.; Denis, Melvin M.; McIntyre, Thomas M.; Weyrich, Andrew S.; Zimmerman, Guy A.

2004-05-01

The mechanisms by which neutrophils, key effector cells of the innate immune system, express new gene products in inflammation are largely uncharacterized. We found that they rapidly translate constitutive mRNAs when activated, a previously unrecognized response. One of the proteins synthesized without a requirement for transcription is the soluble IL-6 receptor , which translocates to endothelial cells and induces a temporal switch to mononuclear leukocyte recruitment. Its synthesis is regulated by a specialized translational control pathway that is inhibited by rapamycin, a bacterial macrolide with therapeutic efficacy in transplantation, inflammatory syndromes, and neoplasia. Signal-dependent translation in activated neutrophils may be a critical mechanism for alteration of the inflammatory milieu and a therapeutic target.

Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ

PubMed Central

Lin, Ying-Hsi; Warren, Chad M.; Li, Jieli; McKinsey, Timothy A.; Russell, Brenda

2016-01-01

The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCε (dnPKCε) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

PubMed

Wojtczak, Blazej A; Sikorski, Pawel J; Fac-Dabrowska, Kaja; Nowicka, Anna; Warminski, Marcin; Kubacka, Dorota; Nowak, Elzbieta; Nowotny, Marcin; Kowalska, Joanna; Jemielity, Jacek

2018-05-09

The 5' cap consists of 7-methylguanosine (m 7 G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m 7 GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL

Efficient pH Dependent Drug Delivery to Target Cancer Cells by Gold Nanoparticles Capped with Carboxymethyl Chitosan

PubMed Central

Madhusudhan, Alle; Reddy, Gangapuram Bhagavanth; Venkatesham, Maragoni; Veerabhadram, Guttena; Kumar, Dudde Anil; Natarajan, Sumathi; Yang, Ming-Yeh; Hu, Anren; Singh, Surya S.

2014-01-01

Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer. PMID:24821542

A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation.

PubMed

Prabhu, S; Saadat, D; Zhang, M; Halbur, L; Fruehauf, J P; Ong, S T

2007-02-22

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

PubMed

Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

2017-01-01

A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

A dormant internal ribosome entry site controls translation of feline immunodeficiency virus.

PubMed

Camerini, Valentina; Decimo, Didier; Balvay, Laurent; Pistello, Mauro; Bendinelli, Mauro; Darlix, Jean-Luc; Ohlmann, Théophile

2008-04-01

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5' untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5' UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a "dormant" IRES that becomes activated by viral infection and cellular stress.

A Dormant Internal Ribosome Entry Site Controls Translation of Feline Immunodeficiency Virus▿

PubMed Central

Camerini, Valentina; Decimo, Didier; Balvay, Laurent; Pistello, Mauro; Bendinelli, Mauro; Darlix, Jean-Luc; Ohlmann, Théophile

2008-01-01

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5′ untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5′ UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a “dormant” IRES that becomes activated by viral infection and cellular stress. PMID:18234788

Iodine-131-labeled, transferrin-capped polypyrrole nanoparticles for tumor-targeted synergistic photothermal-radioisotope therapy.

PubMed

Song, Xuejiao; Liang, Chao; Feng, Liangzhu; Yang, Kai; Liu, Zhuang

2017-08-22

Combining different therapeutic functions within single tumor-targeted nanoscale delivery systems is promising to overcome the limitations of conventional cancer therapies. Herein, transferrin that recognizes transferrin receptors up-regulated on tumor cells is pre-labeled with iodine-131 ( 131 I) and then utilized as the stabilizer in the fabrication of polypyrrole (PPy) nanoparticles. The obtained transferrin-capped PPy@Tf- 131 I nanoparticles could be used for tumor-targeted radioisotope therapy (RIT) and photothermal therapy (PTT), by employing beta-emission from 131 I and the intrinsic high near-infrared (NIR) absorbance of PPy, respectively. Owing to the transferrin-mediated tumor targeting, PPy@Tf- 131 I nanoparticles exhibit obviously enhanced in vitro cancer cell binding and in vivo tumor uptake compared to its non-targeting counterpart. The combined RIT and PTT based on PPy@Tf- 131 I nanoparticles is then conducted, achieving a remarkable synergistic therapeutic effect. This work thus demonstrates a rather simple one-step approach to fabricate tumor-targeting nanoparticles based on protein-capped conjugated polymers, promising for combination cancer therapy with great efficacy and high safety.

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

PubMed

Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A; Ravindranath, Vijayalakshmi

2017-12-01

Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APP Swe /PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

Dynamic landscape of the local translation at activated synapses.

PubMed

Khlebodarova, T M; Kogai, V V; Trifonova, E A; Likhoshvai, V A

2018-01-01

The mammalian target of rapamycin (mTOR) signaling pathway is the central regulator of cap-dependent translation at the synapse. Disturbances in mTOR pathway have been associated with several neurological diseases, such as autism and epilepsy. RNA-binding protein FMRP, a negative regulator of translation initiation, is one of the key components of the local translation system. Activation and inactivation of FMRP occurs via phosphorylation by S6 kinase and dephosphorylation by PP2A phosphatase, respectively. S6 kinase and PP2A phosphatase are activated in response to mGluR receptor stimulation through different signaling pathways and at different rates. The dynamic aspects of this system are poorly understood. We developed a mathematical model of FMRP-dependent regulation of postsynaptic density (PSD) protein synthesis in response to mGluR receptor stimulation and conducted in silico experiments to study the regulatory circuit functioning. The modeling results revealed the possibility of generating oscillatory (cyclic and quasi-cyclic), chaotic and even hyperchaotic dynamics of postsynaptic protein synthesis as well as the presence of multiple attractors in a wide range of parameters of the local translation system. The results suggest that autistic disorders associated with mTOR pathway hyperactivation may be due to impaired proteome stability associated with the formation of complex dynamic regimes of PSD protein synthesis in response to stimulation of mGluR receptors on the postsynaptic membrane of excitatory synapses on pyramidal hippocampal cells.

Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sally A. Mackenzie

2011-04-18

A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility thatmore » had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.« less

Immediate Translation of Formin DIAPH1 mRNA after Its Exiting the Nucleus Is Required for Its Perinuclear Localization in Fibroblasts

PubMed Central

Liao, Guoning; Liu, Gang

2013-01-01

DIAPH1 is a formin protein which promotes actin polymerization, stabilizes microtubules and consequently is involved in cytoskeleton dynamics, cell migration and differentiation. In contrast to the relatively well-understood signaling cascades that regulate DIAPH1 activity, its spatial regulation of biogenesis is not understood. A recent report showed that synthesis of DIAPH1 is confined in the perinuclear ER compartment through translation-dependent mRNA targeting. However, the underlying mechanism of DIAPH1 local synthesis is yet to be elucidated. Here, we provide evidence to demonstrate that the 5′-cap-mediated immediate translation of DIAPH1 mRNA upon exiting nucleus is required for localizing the mRNA in the perinuclear ER compartment. This is supported by data: 1) Delayed translation of DIAPH1 mRNA resulted in loss of perinuclear localization of the mRNA; 2) Once delocalized, DIAPH1 mRNA could not be retargeted to the perinuclear region; and 3) The translation of DIAPH1 mRNA is 5′-cap dependent. These results provide new insights into the novel mechanism of DIAPH1 local synthesis. In addition, these findings have led to the development of new approaches for manipulating DIAPH1 mRNA localization and local protein synthesis in cells for functional studies. Furthermore, a correlation of DIAPH1 mRNA and DIAPH1 protein localization has been demonstrated using a new method to quantify the intracellular distribution of protein. PMID:23840831

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease

PubMed Central

Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A.

2017-01-01

Abstract Aims: Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Results: Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APPSwe/PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9–10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Innovation: Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. Conclusions: We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269–1280. PMID:28264587

mRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

PubMed Central

Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick

2012-01-01

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m7GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5′cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation

PubMed Central

Anderson, Grace R.; Wardell, Suzanne E.; Cakir, Merve; Crawford, Lorin; Leeds, Jim C.; Nussbaum, Daniel P.; Shankar, Pallavi S.; Soderquist, Ryan S.; Stein, Elizabeth M.; Tingley, Jennifer P.; Winter, Peter S.; Zieser-Misenheimer, Elizabeth K.; Alley, Holly M.; Yllanes, Alexander; Haney, Victoria; Blackwell, Kimberly L.; McCall, Shannon J.; McDonnell, Donald P.; Wood, Kris C.

2017-01-01

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that the combined inhibition of BCL-XL and the mTOR/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses MCL-1 protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-XL. This dual dependence on BCL-XL and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of molecular subtype or PIK3CA mutational status. Further, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-XL/MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to anti-apoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-XL also sensitizes breast cancer cells to standard of care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-XL and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-XL may be effective in tumors lacking PIK3CA mutations. PMID:27974663

ALCOHOLIC HEPATITIS: TRANSLATIONAL APPROACHES TO DEVELOP TARGETED THERAPIES

PubMed Central

Mandrekar, Pranoti; Bataller, Ramon; Tsukamoto, Hidekazu; Gao, Bin

2016-01-01

Alcoholic liver disease (ALD) is a leading cause of liver related mortality worldwide. In contrast to recent advances in therapeutic strategies for patients with viral hepatitis, there is a significant lack of novel therapeutic options for patients with ALD. In particular, there is an urgent need to focus our efforts on effective therapeutic interventions for alcoholic hepatitis (AH), the most severe form of ALD. AH is characterized by an abrupt development of jaundice and complications related to liver insufficiency and portal hypertension in patients with heavy alcohol intake. The mortality of patients with AH is very high (20–50% at 3 months). Available therapies are not effective in many patients and targeted approaches are imminently needed. The development of such therapies requires translational studies in human samples and suitable animal models that reproduce clinical and histological features of AH. In recent years, new animal models that simulate some of the features of human AH have been developed, and translational studies using human samples have identified potential pathogenic factors and histological parameters that predict survival. This review article summarizes the unmet needs for translational studies on the pathogenesis of AH, pre-clinical translational tools, and emerging drug targets to benefit the AH patient. PMID:26940353

Targeting interleukin-1β in CAPS (cryopyrin-associated periodic) syndromes: what did we learn?

PubMed

Koné-Paut, Isabelle; Piram, Maryam

2012-11-01

CAPS is the prototype of an IL-1β driven auto inflammatory disorder. Features of recurrent systemic inflammation compromises patient's quality of life, and may reduce life expectancy by inducing definite organ damage. Recent treatment targeting IL-1 have shown dramatic effect on patient's clinical symptoms and quality of life. Anakinra was first used successfully in treating small series of patients with all CAPS phenotypes. Two pivotal randomized placebo-controlled studies allowed licensing of rilonacept and canakinumab as orphan drugs for CAPS patients. The use of anti-IL-1 drugs in CAPS is still relatively new, and their effect on a long term is not well known. As we can suppress the clinical symptoms of patients with CAPS, important questions remain regarding the full effect of anti-IL-1 treatment on organ involvement and their potential ability to prevent them. As important variations of treatment doses and schedules appear in reaching effectiveness, pharmacologic studies are still warranted to determine a potential diffusion of anti-IL-1 drugs in the fluids and tissues. More studies evaluating the efficacy and safety of anti-IL-1 drugs given in patients before 2 years of age are warranted, since it is believed that the earliest treatment could avoid secondary CAPS complications to develop. Copyright © 2012 Elsevier B.V. All rights reserved.

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

NASA Astrophysics Data System (ADS)

Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

2014-09-01

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

The Frustrated Host Response to Legionella pneumophila Is Bypassed by MyD88-Dependent Translation of Pro-inflammatory Cytokines

PubMed Central

Asrat, Seblewongel; Dugan, Aisling S.; Isberg, Ralph R.

2014-01-01

Many pathogens, particularly those that require their host for survival, have devised mechanisms to subvert the host immune response in order to survive and replicate intracellularly. Legionella pneumophila, the causative agent of Legionnaires' disease, promotes intracellular growth by translocating proteins into its host cytosol through its type IV protein secretion machinery. At least 5 of the bacterial translocated effectors interfere with the function of host cell elongation factors, blocking translation and causing the induction of a unique host cell transcriptional profile. In addition, L. pneumophila also interferes with translation initiation, by preventing cap-dependent translation in host cells. We demonstrate here that protein translation inhibition by L. pneumophila leads to a frustrated host MAP kinase response, where genes involved in the pathway are transcribed but fail to be translated due to the bacterium-induced protein synthesis inhibition. Surprisingly, few pro-inflammatory cytokines, such as IL-1α and IL-1β, bypass this inhibition and get synthesized in the presence of Legionella effectors. We show that the selective synthesis of these genes requires MyD88 signaling and takes place in both infected cells that harbor bacteria and neighboring bystander cells. Our findings offer a perspective of how host cells are able to cope with pathogen-encoded activities that disrupt normal cellular process and initiate a successful inflammatory response. PMID:25058342

'A unique 5’ translation element discoverd in Triticum mosaic virus'

USDA-ARS?s Scientific Manuscript database

Many RNA viruses rely on internal ribosome entry site (IRES) elements to deviate from the canonical cap-dependent translation mechanism. In contrast to the well-defined IRES elements found in animal viruses, plant viral IRESes identified to date reportedly consist of relatively short, ill-defined se...

Alcoholic hepatitis: Translational approaches to develop targeted therapies.

PubMed

Mandrekar, Pranoti; Bataller, Ramon; Tsukamoto, Hidekazu; Gao, Bin

2016-10-01

Alcoholic liver disease is a leading cause of liver-related mortality worldwide. In contrast to recent advances in therapeutic strategies for patients with viral hepatitis, there is a significant lack of novel therapeutic options for patients with alcoholic liver disease. In particular, there is an urgent need to focus our efforts on effective therapeutic interventions for alcoholic hepatitis (AH), the most severe form of alcoholic liver disease. AH is characterized by an abrupt development of jaundice and complications related to liver insufficiency and portal hypertension in patients with heavy alcohol intake. The mortality of patients with AH is very high (20%-50% at 3 months). Available therapies are not effective in many patients, and targeted approaches are imminently needed. The development of such therapies requires translational studies in human samples and suitable animal models that reproduce the clinical and histological features of AH. In recent years, new animal models that simulate some of the features of human AH have been developed, and translational studies using human samples have identified potential pathogenic factors and histological parameters that predict survival. This review summarizes the unmet needs for translational studies on the pathogenesis of AH, preclinical translational tools, and emerging drug targets to benefit the AH patient. (Hepatology 2016;64:1343-1355). © 2016 by the American Association for the Study of Liver Diseases.

Melanoma-Targeted Chemothermotherapy and In Situ Peptide Immunotherapy through HSP Production by Using Melanogenesis Substrate, NPrCAP, and Magnetite Nanoparticles

PubMed Central

Jimbow, Kowichi; Ishii-Osai, Yasue; Ito, Shosuke; Tamura, Yasuaki; Ito, Akira; Yoneta, Akihiro; Kamiya, Takafumi; Yamashita, Toshiharu; Honda, Hiroyuki; Wakamatsu, Kazumasa; Murase, Katsutoshi; Nohara, Satoshi; Nakayama, Eiichi; Hasegawa, Takeo; Yamamoto, Itsuo; Kobayashi, Takeshi

2013-01-01

Exploitation of biological properties unique to cancer cells may provide a novel approach to overcome difficult challenges to the treatment of advanced melanoma. In order to develop melanoma-targeted chemothermoimmunotherapy, a melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP), sulfur-amine analogue of tyrosine, was conjugated with magnetite nanoparticles. NPrCAP was exploited from melanogenesis substrates, which are expected to be selectively incorporated into melanoma cells and produce highly reactive free radicals through reacting with tyrosinase, resulting in chemotherapeutic and immunotherapeutic effects by oxidative stress and apoptotic cell death. Magnetite nanoparticles were conjugated with NPrCAP to introduce thermotherapeutic and immunotherapeutic effects through nonapoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). During these therapeutic processes, NPrCAP was also expected to provide melanoma-targeted drug delivery system. PMID:23533767

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency*

PubMed Central

Banerjee, Bidisha; Goss, Dixie J.

2014-01-01

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3′-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3′ BTE with higher affinity than for either m7G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3′ BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

PubMed

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2018-03-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

PubMed

Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

2010-04-15

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

International Test Comparisons: Reviewing Translation Error in Different Source Language-Target Language Combinations

ERIC Educational Resources Information Center

Zhao, Xueyu; Solano-Flores, Guillermo; Qian, Ming

2018-01-01

This article addresses test translation review in international test comparisons. We investigated the applicability of the theory of test translation error--a theory of the multidimensionality and inevitability of test translation error--across source language-target language combinations in the translation of PISA (Programme of International…

Target-responsive DNA-capped nanocontainer used for fabricating universal detector and performing logic operations

PubMed Central

Wu, Li; Ren, Jinsong; Qu, Xiaogang

2014-01-01

Nucleic acids have become a powerful tool in nanotechnology because of their controllable diverse conformational transitions and adaptable higher-order nanostructure. Using single-stranded DNA probes as the pore-caps for various target recognition, here we present an ultrasensitive universal electrochemical detection system based on graphene and mesoporous silica, and achieve sensitivity with all of the major classes of analytes and simultaneously realize DNA logic gate operations. The concept is based on the locking of the pores and preventing the signal-reporter molecules from escape by target-induced the conformational change of the tailored DNA caps. The coupling of ‘waking up’ gatekeeper with highly specific biochemical recognition is an innovative strategy for the detection of various targets, able to compete with classical methods which need expensive instrumentation and sophisticated experimental operations. The present study has introduced a new electrochemical signal amplification concept and also adds a new dimension to the function of graphene-mesoporous materials hybrids as multifunctional nanoscale logic devices. More importantly, the development of this approach would spur further advances in important areas, such as point-of-care diagnostics or detection of specific biological contaminations, and hold promise for use in field analysis. PMID:25249622

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

PubMed

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation

PubMed Central

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

PubMed

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

2017-06-01

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Protein synthesis in geostimulated root caps

NASA Technical Reports Server (NTRS)

Feldman, L. J.

1982-01-01

A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

PubMed

de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

2013-09-27

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

PubMed

Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

2014-06-01

The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

Residual translation compensations in radar target narrowband imaging based on trajectory information

NASA Astrophysics Data System (ADS)

Yue, Wenjue; Peng, Bo; Wei, Xizhang; Li, Xiang; Liao, Dongping

2018-05-01

High velocity translation will result in defocusing scattering centers in radar imaging. In this paper, we propose a Residual Translation Compensations (RTC) method based on target trajectory information to eliminate the translation effects in radar imaging. Translation could not be simply regarded as a uniformly accelerated motion in reality. So the prior knowledge of the target trajectory is introduced to enhance compensation precision. First we use the two-body orbit model to figure out the radial distance. Then, stepwise compensations are applied to eliminate residual propagation delay based on conjugate multiplication method. Finally, tomography is used to confirm the validity of the method. Compare with translation parameters estimation method based on the spectral peak of the conjugate multiplied signal, RTC method in this paper enjoys a better tomography result. When the Signal Noise Ratio (SNR) of the radar echo signal is 4dB, the scattering centers can also be extracted clearly.

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site.

PubMed

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S; Mkhize, Nonhlanhla N; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D; Labuschagne, Phillip; Louder, Mark K; Bailer, Robert T; Abdool Karim, Salim S; Mascola, John R; Williamson, Carolyn; Moore, Penny L; Kwong, Peter D; Morris, Lynn

2016-11-15

All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report themore » isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

PubMed Central

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

2016-01-01

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site

Antipsychotics Activate mTORC1-Dependent Translation to Enhance Neuronal Morphological Complexity

PubMed Central

Bowling, Heather; Zhang, Guoan; Bhattacharya, Aditi; Pérez-Cuesta, Luis M.; Deinhardt, Katrin; Hoeffer, Charles A.; Neubert, Thomas A.; Gan, Wen-biao; Klann, Eric; Chao, Moses V.

2014-01-01

Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. Here, we showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D2R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D2R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics. PMID:24425786

Isolation of tungsten and tantalum isotopes without supports from. cap alpha. -particle-irradiated hafnium targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gasita, S.M.; Iota, B.Z.; Malachkov, A.G.

1985-11-01

An extraction procedure has been developed for successive isolation of tungsten (/sup 178/W and /sup 181/W) and tantalum (/sup 179/Ta and /sup 182/Ta) isotopes without supports from ..cap alpha..particle-irradiated hafnium targets. The target, irradiated on a cyclotron, is dissolved in hydrofluoric acid. Tantalum isotopes are extracted with tributyl phosphate (TBP) from 1-5 M HF and are then reextracted with a 1:1 ammonia solution, and hydrofluoric acid is removed by heating. Tungsten isotopes are extracted with a chloroform solution or N-benzoyl-N-phenylhydroxylamine (BPHA) from 11-12 M H/sub 2/SO/sub 4/ or ..cap alpha..-benzoin oxime from 4.5-5.5 M H/sub 2/SO/sub 4/ and are thenmore » reextracted with a l:l ammonia solution. The yield of tungsten isotopes is not less than 95%, and the content of radioactive impurities of other isotopes is not more than 0.1%.« less

Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

PubMed Central

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2017-01-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424

Targeted presurgical decompensation in patients with yaw-dependent facial asymmetry

PubMed Central

Kim, Kyung-A; Lee, Ji-Won; Park, Jeong-Ho; Kim, Byoung-Ho; Ahn, Hyo-Won

2017-01-01

Facial asymmetry can be classified into the rolling-dominant type (R-type), translation-dominant type (T-type), yawing-dominant type (Y-type), and atypical type (A-type) based on the distorted skeletal components that cause canting, translation, and yawing of the maxilla and/or mandible. Each facial asymmetry type represents dentoalveolar compensations in three dimensions that correspond to the main skeletal discrepancies. To obtain sufficient surgical correction, it is necessary to analyze the main skeletal discrepancies contributing to the facial asymmetry and then the skeletal-dental relationships in the maxilla and mandible separately. Particularly in cases of facial asymmetry accompanied by mandibular yawing, it is not simple to establish pre-surgical goals of tooth movement since chin deviation and posterior gonial prominence can be either aggravated or compromised according to the direction of mandibular yawing. Thus, strategic dentoalveolar decompensations targeting the real basal skeletal discrepancies should be performed during presurgical orthodontic treatment to allow for sufficient skeletal correction with stability. In this report, we document targeted decompensation of two asymmetry patients focusing on more complicated yaw-dependent types than others: Y-type and A-type. This may suggest a clinical guideline on the targeted decompensation in patient with different types of facial asymmetries. PMID:28523246

Targeted presurgical decompensation in patients with yaw-dependent facial asymmetry.

PubMed

Kim, Kyung-A; Lee, Ji-Won; Park, Jeong-Ho; Kim, Byoung-Ho; Ahn, Hyo-Won; Kim, Su-Jung

2017-05-01

Facial asymmetry can be classified into the rolling-dominant type (R-type), translation-dominant type (T-type), yawing-dominant type (Y-type), and atypical type (A-type) based on the distorted skeletal components that cause canting, translation, and yawing of the maxilla and/or mandible. Each facial asymmetry type represents dentoalveolar compensations in three dimensions that correspond to the main skeletal discrepancies. To obtain sufficient surgical correction, it is necessary to analyze the main skeletal discrepancies contributing to the facial asymmetry and then the skeletal-dental relationships in the maxilla and mandible separately. Particularly in cases of facial asymmetry accompanied by mandibular yawing, it is not simple to establish pre-surgical goals of tooth movement since chin deviation and posterior gonial prominence can be either aggravated or compromised according to the direction of mandibular yawing. Thus, strategic dentoalveolar decompensations targeting the real basal skeletal discrepancies should be performed during presurgical orthodontic treatment to allow for sufficient skeletal correction with stability. In this report, we document targeted decompensation of two asymmetry patients focusing on more complicated yaw-dependent types than others: Y-type and A-type. This may suggest a clinical guideline on the targeted decompensation in patient with different types of facial asymmetries.

Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane*♦

PubMed Central

de Oliveira, Guilherme A. P.; Pereira, Elen G.; Ferretti, Giulia D. S.; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L.

2013-01-01

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl+ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target. PMID:23928308

Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation

PubMed Central

Tomlinson, Gareth H.; Miles, Katherine; Smith, Richard W. P.; Rossi, Adriano G.; Hiemstra, Pieter S.; van ’t Wout, Emily F. A.; Dean, Jonathan L. E.; Gray, Nicola K.; Lu, Wuyuan; Gray, Mohini

2016-01-01

Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection with Salmonella enterica serovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a “molecular brake” on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage. PMID:27044108

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

DOE PAGES

Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; ...

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitorymore » mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.« less

MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

PubMed Central

Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

2016-01-01

MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

PubMed

Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley.

PubMed

Khandal, Dhriti; Samol, Iga; Buhr, Frank; Pollmann, Stephan; Schmidt, Holger; Clemens, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

2009-08-04

The tigrina (tig)-d.12 mutant of barley is impaired in the negative control limiting excess protochlorophyllide (Pchlide) accumulation in the dark. Upon illumination, Pchlide operates as photosensitizer and triggers singlet oxygen production and cell death. Here, we show that both Pchlide and singlet oxygen operate as signals that control gene expression and metabolite accumulation in tig-d.12 plants. In vivo labeling, Northern blotting, polysome profiling, and protein gel blot analyses revealed a selective suppression of synthesis of the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCSs and RBCLs), the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCB2), as well as other chlorophyll-binding proteins, in response to singlet oxygen. In part, these effects were caused by an arrest in translation initiation of photosynthetic transcripts at 80S cytoplasmic ribosomes. The observed changes in translation correlated with a decline in the phosphorylation level of ribosomal protein S6. At later stages, ribosome dissociation occurred. Together, our results identify translation as a major target of singlet oxygen-dependent growth control and cell death in higher plants.

Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley

PubMed Central

Khandal, Dhriti; Samol, Iga; Buhr, Frank; Pollmann, Stephan; Schmidt, Holger; Clemens, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

2009-01-01

The tigrina (tig)-d.12 mutant of barley is impaired in the negative control limiting excess protochlorophyllide (Pchlide) accumulation in the dark. Upon illumination, Pchlide operates as photosensitizer and triggers singlet oxygen production and cell death. Here, we show that both Pchlide and singlet oxygen operate as signals that control gene expression and metabolite accumulation in tig-d.12 plants. In vivo labeling, Northern blotting, polysome profiling, and protein gel blot analyses revealed a selective suppression of synthesis of the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCSs and RBCLs), the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCB2), as well as other chlorophyll-binding proteins, in response to singlet oxygen. In part, these effects were caused by an arrest in translation initiation of photosynthetic transcripts at 80S cytoplasmic ribosomes. The observed changes in translation correlated with a decline in the phosphorylation level of ribosomal protein S6. At later stages, ribosome dissociation occurred. Together, our results identify translation as a major target of singlet oxygen-dependent growth control and cell death in higher plants. PMID:19620736

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

PubMed Central

Khaperskyy, Denys A.; Emara, Mohamed M.; Johnston, Benjamin P.; Anderson, Paul; Hatchette, Todd F.; McCormick, Craig

2014-01-01

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication. PMID:25010204

Glutamate-dependent phosphorylation of the mammalian target of rapamycin (mTOR) in Bergmann glial cells.

PubMed

Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Suárez-Pozos, Edna; Melgarejo, Yaaziel; González-Mejia, Elba; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Aguilera, José; Ortega, Arturo

2009-09-01

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, plays an important role in neuronal development and synaptic plasticity. It activates a variety of signaling pathways that regulate gene expression at the transcriptional and translational levels. Within glial cells, besides transcription, glutamate also regulates translation initiation and elongation. The mammalian target of rapamycin (mTOR), a key participant in the translation process, represents an important regulatory locus for translational control. Therefore, in the present communication we sought to characterize the mTOR phosphorylation pattern after glutamate treatment in chick cerebellar Bergmann glia primary cultures. A time- and dose-dependent increase in mTOR Ser 2448 phosphorylation was found. Pharmacological tools established that the glutamate effect is mediated through ionotropic and metabotropic receptors and interestingly, the glutamate transporter system is also involved. The signaling cascade triggered by glutamate includes an increase in intracellular Ca2+ levels, and the activation of the p60(Src)/PI-3K/PKB pathway. These results suggest that glia cells participate in the activity-dependent change in the brain protein repertoire.

mTORC1-dependent translation of collapsin response mediator protein-2 drives neuroadaptations underlying excessive alcohol-drinking behaviors

PubMed Central

Liu, F; Laguesse, S; Legastelois, R; Morisot, N; Ben Hamida, S; Ron, D

2017-01-01

Mammalian target of rapamycin complex 1 (mTORC1) has an essential role in dendritic mRNA translation and participates in mechanisms underlying alcohol-drinking and reconsolidation of alcohol-related memories. Here, we report that excessive alcohol consumption increases the translation of downstream targets of mTORC1, including collapsin response mediator protein-2 (CRMP-2), in the nucleus accumbens (NAc) of rodents. We show that alcohol-mediated induction of CRMP-2 translation is mTORC1-dependent, leading to increased CRMP-2 protein levels. Furthermore, we demonstrate that alcohol intake also blocks glycogen synthase kinase-3β (GSK-3β)-phosphorylation of CRMP-2, which results in elevated binding of CRMP-2 to microtubules and a concomitant increase in microtubule content. Finally, we show that systemic administration of the CRMP-2 inhibitor lacosamide, or knockdown of CRMP-2 in the NAc decreases excessive alcohol intake. These results suggest that CRMP-2 in the NAc is a convergent point that receives inputs from two signaling pathways, mTORC1 and GSK-3β, that in turn drives excessive alcohol-drinking behaviors. PMID:26952865

'2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

PubMed

Roulston, Claire; Luke, Garry A; de Felipe, Pablo; Ruan, Lin; Cope, Jonathan; Nicholson, John; Sukhodub, Andriy; Tilsner, Jens; Ryan, Martin D

2016-08-01

We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting. © 2016 The Authors. Traffic published by John Wiley & Sons Ltd.

Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states.

PubMed

Komar, Anton A; Hatzoglou, Maria

2011-01-15

Translation of cellular mRNAs via initiation at Internal Ribosome Entry Sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

PubMed Central

Mahe, Karan; Ou, Tingyoung; Castro, Noemi M; Christensen, Lana N; Cheung, Lee; Jiang, Xueer; Yoon, Daniel; Huang, Bo

2018-01-01

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism. PMID:29708497

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies.

PubMed

Deng, Changchun; Lipstein, Mark R; Scotto, Luigi; Jirau Serrano, Xavier O; Mangone, Michael A; Li, Shirong; Vendome, Jeremie; Hao, Yun; Xu, Xiaoming; Deng, Shi-Xian; Realubit, Ronald B; Tatonetti, Nicholas P; Karan, Charles; Lentzsch, Suzanne; Fruman, David A; Honig, Barry; Landry, Donald W; O'Connor, Owen A

2017-01-05

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. © 2017 by The American Society of Hematology.

Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function

PubMed Central

Mullen, Nicholas J.

2017-01-01

Capping of nascent RNA polymerase II (Pol II) transcripts is required for gene expression and the first two steps are catalyzed by separate 5′ triphosphatase and guanylyltransferase activities of the human capping enzyme (HCE). The cap is added co-transcriptionally, but how the two activities are coordinated is unclear. Our previous in vitro work has suggested that an unidentified factor modulates the minimum length at which nascent transcripts can be capped. Using the same well-established in vitro system with hydrogen peroxide as a capping inhibitor, we show that this unidentified factor targets the guanylyltransferase activity of HCE. We also uncover the mechanism of HCE inhibition by hydrogen peroxide, and by using mass spectrometry demonstrate that the active site cysteine residue of the HCE triphosphatase domain becomes oxidized. Using recombinant proteins for the two separated HCE domains, we provide evidence that the triphosphatase normally acts on transcripts shorter than can be acted upon by the guanylyltransferase. Our further characterization of the capping reaction dependence on transcript length and its interaction with the unidentified modulator of capping raises the interesting possibility that the capping reaction could be regulated. PMID:29028835

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

PubMed Central

Laudenbach, Beatrice Theres; Martínez-Montero, Saúl; Cencic, Regina; Habjan, Matthias; Pichlmair, Andreas; Damha, Masad J.; Pelletier, Jerry; Nagar, Bhushan

2017-01-01

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2′-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2′-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA. PMID:28251928

Cell fate determination by ubiquitin-dependent regulation of translation

PubMed Central

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

2015-01-01

Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

West nile virus-induced activation of mammalian target of rapamycin complex 1 supports viral growth and viral protein expression.

PubMed

Shives, Katherine D; Beatman, Erica L; Chamanian, Mastooreh; O'Brien, Caitlin; Hobson-Peters, Jody; Beckham, J David

2014-08-01

Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth. Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states in the United States and is now the leading cause of epidemic encephalitis in North America. Currently, the mechanism by which flaviviruses such as WNV translate their genomes in

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

PubMed

Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Targeting neutrophils in ischemic stroke: translational insights from experimental studies

PubMed Central

Jickling, Glen C; Liu, DaZhi; Ander, Bradley P; Stamova, Boryana; Zhan, Xinhua; Sharp, Frank R

2015-01-01

Neutrophils have key roles in ischemic brain injury, thrombosis, and atherosclerosis. As such, neutrophils are of great interest as targets to treat and prevent ischemic stroke. After stroke, neutrophils respond rapidly promoting blood–brain barrier disruption, cerebral edema, and brain injury. A surge of neutrophil-derived reactive oxygen species, proteases, and cytokines are released as neutrophils interact with cerebral endothelium. Neutrophils also are linked to the major processes that cause ischemic stroke, thrombosis, and atherosclerosis. Thrombosis is promoted through interactions with platelets, clotting factors, and release of prothrombotic molecules. In atherosclerosis, neutrophils promote plaque formation and rupture by generating oxidized-low density lipoprotein, enhancing monocyte infiltration, and degrading the fibrous cap. In experimental studies targeting neutrophils can improve stroke. However, early human studies have been met with challenges, and suggest that selective targeting of neutrophils may be required. Several properties of neutrophil are beneficial and thus may important to preserve in patients with stroke including antimicrobial, antiinflammatory, and neuroprotective functions. PMID:25806703

Targeting inflammation in pancreatic cancer: Clinical translation

PubMed Central

Steele, Colin William; Kaur Gill, Nina Angharad; Jamieson, Nigel Balfour; Carter, Christopher Ross

2016-01-01

Preclinical modelling studies are beginning to aid development of therapies targeted against key regulators of pancreatic cancer progression. Pancreatic cancer is an aggressive, stromally-rich tumor, from which few people survive. Within the tumor microenvironment cellular and extracellular components exist, shielding tumor cells from immune cell clearance, and chemotherapy, enhancing progression of the disease. The cellular component of this microenvironment consists mainly of stellate cells and inflammatory cells. New findings suggest that manipulation of the cellular component of the tumor microenvironment is possible to promote immune cell killing of tumor cells. Here we explore possible immunogenic therapeutic strategies. Additionally extracellular stromal elements play a key role in protecting tumor cells from chemotherapies targeted at the pancreas. We describe the experimental findings and the pitfalls associated with translation of stromally targeted therapies to clinical trial. Finally, we discuss the key inflammatory signal transducers activated subsequent to driver mutations in oncogenic Kras in pancreatic cancer. We present the preclinical findings that have led to successful early trials of STAT3 inhibitors in pancreatic adenocarcinoma. PMID:27096033

Protein and carotenoid synthesis and turnover in gravistimulated root caps

NASA Technical Reports Server (NTRS)

Feldman, L. J.

1984-01-01

In certain cultivars of corn gravitropic bending occurs only after the root cap, the site of gravity perception, is exposed to light. Light appears to trigger or to remove some block in the gravity translation process. Using light sensitive cultivars of corn, it was shown that light affects various processes in the cap. The roles of these light-induced processes in gravitropic bending in roots were studied.

Binary translation using peephole translation rules

DOEpatents

Bansal, Sorav; Aiken, Alex

2010-05-04

An efficient binary translator uses peephole translation rules to directly translate executable code from one instruction set to another. In a preferred embodiment, the translation rules are generated using superoptimization techniques that enable the translator to automatically learn translation rules for translating code from the source to target instruction set architecture.

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonenberg, N.; Guertin, D.; Lee, K.A.W.

1982-12-01

Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

Translation-coupling systems

DOEpatents

Pfleger, Brian; Mendez-Perez, Daniel

2013-11-05

Disclosed are systems and methods for coupling translation of a target gene to a detectable response gene. A version of the invention includes a translation-coupling cassette. The translation-coupling cassette includes a target gene, a response gene, a response-gene translation control element, and a secondary structure-forming sequence that reversibly forms a secondary structure masking the response-gene translation control element. Masking of the response-gene translation control element inhibits translation of the response gene. Full translation of the target gene results in unfolding of the secondary structure and consequent translation of the response gene. Translation of the target gene is determined by detecting presence of the response-gene protein product. The invention further includes RNA transcripts of the translation-coupling cassettes, vectors comprising the translation-coupling cassettes, hosts comprising the translation-coupling cassettes, methods of using the translation-coupling cassettes, and gene products produced with the translation-coupling cassettes.

Translation-coupling systems

DOEpatents

Pfleger, Brian; Mendez-Perez, Daniel

2015-05-19

Disclosed are systems and methods for coupling translation of a target gene to a detectable response gene. A version of the invention includes a translation-coupling cassette. The translation-coupling cassette includes a target gene, a response gene, a response-gene translation control element, and a secondary structure-forming sequence that reversibly forms a secondary structure masking the response-gene translation control element. Masking of the response-gene translation control element inhibits translation of the response gene. Full translation of the target gene results in unfolding of the secondary structure and consequent translation of the response gene. Translation of the target gene is determined by detecting presence of the response-gene protein product. The invention further includes RNA transcripts of the translation-coupling cassettes, vectors comprising the translation-coupling cassettes, hosts comprising the translation-coupling cassettes, methods of using the translation-coupling cassettes, and gene products produced with the translation-coupling cassettes.

Multifunctional polymer-capped mesoporous silica nanoparticles for pH-responsive targeted drug delivery.

PubMed

Niedermayer, Stefan; Weiss, Veronika; Herrmann, Annika; Schmidt, Alexandra; Datz, Stefan; Müller, Katharina; Wagner, Ernst; Bein, Thomas; Bräuchle, Christoph

2015-05-07

A highly stable modular platform, based on the sequential covalent attachment of different functionalities to the surface of core-shell mesoporous silica nanoparticles (MSNs) for targeted drug delivery is presented. A reversible pH-responsive cap system based on covalently attached poly(2-vinylpyridine) (PVP) was developed as drug release mechanism. Our platform offers (i) tuneable interactions and release kinetics with the cargo drug in the mesopores based on chemically orthogonal core-shell design, (ii) an extremely robust and reversible closure and release mechanism based on endosomal acidification of the covalently attached PVP polymer block, (iii) high colloidal stability due to a covalently coupled PEG shell, and (iv) the ability to covalently attach a wide variety of dyes, targeting ligands and other functionalities at the outer periphery of the PEG shell. The functionality of the system was demonstrated in several cell studies, showing pH-triggered release in the endosome, light-triggered endosomal escape with an on-board photosensitizer, and efficient folic acid-based cell targeting.

Cell-fate determination by ubiquitin-dependent regulation of translation.

PubMed

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T; Rape, Michael

2015-09-24

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Translation compensation and micro-Doppler extraction for precession ballistic targets with a wideband terahertz radar

NASA Astrophysics Data System (ADS)

Yang, Qi; Deng, Bin; Wang, Hongqiang; Zhang, Ye; Qin, Yuliang

2018-01-01

Imaging, classification, and recognition techniques of ballistic targets in midcourse have always been the focus of research in the radar field for military applications. However, the high velocity translation of ballistic targets will subject range profile and Doppler to translation, slope, and fold, which are especially severe in the terahertz region. Therefore, a two-step translation compensation method based on envelope alignment is presented. The rough compensation is based on the traditional envelope alignment algorithm in inverse synthetic aperture radar imaging, and the fine compensation is supported by distance fitting. Then, a wideband imaging radar system with a carrier frequency of 0.32 THz is introduced, and an experiment on a precession missile model is carried out. After translation compensation with the method proposed in this paper, the range profile and the micro-Doppler distributions unaffected by translation are obtained, providing an important foundation for the high-resolution imaging and micro-Doppler extraction of the terahertz radar.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations.

PubMed

Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng

2014-11-28

A universal molecular translator based on the target-triggered DNA strand displacement was developed, which was able to convert various kinds of non-nucleic acid targets into a unique output DNA. This translation strategy was successfully applied in directing dynamic DNA assemblies and in realizing three-input logic gate operations.

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts.

PubMed Central

Cowsert, L M; Fox, M C; Zon, G; Mirabelli, C K

1993-01-01

Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11. Images PMID:8383937

Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

PubMed Central

Iliff, Adam J.; Renoux, Abigail J.; Krans, Amy; Usdin, Karen; Sutton, Michael A.; Todd, Peter K.

2013-01-01

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5′ untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120–150 CGG repeats in the mouse Fmr1 5′ UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3–CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity. PMID:23250915

Milk—A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation

PubMed Central

Melnik, Bodo C.

2015-01-01

Based on own translational research of the biochemical and hormonal effects of cow’s milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1) essential branched-chain amino acids (BCAAs); (2) glutamine; (3) palmitic acid; and (4) bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER) stress and drives an aimless quasi-program, which promotes aging and age-related diseases. PMID:26225961

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

PubMed

Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

2018-04-01

Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl 2 , up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Translating the semi-structured assessment for drug dependence and alcoholism in the Western Pacific: rationale, study design and reliability of alcohol dependence.

PubMed

Quinn, Amity E; Rosen, Rochelle K; McGeary, John E; Amoa, Francine; Kranzler, Henry R; Francazio, Sarah; McGarvey, Stephen T; Swift, Robert M

2014-01-01

The aims of this study were to develop a bilingual version of the Semi-Structured Assessment for Drug Dependence and Alcoholism (SSADDA) in English and Samoan and determine the reliability of assessments of alcohol dependence in American Samoa. The study consisted of development and reliability-testing phases. In the development phase, the SSADDA alcohol module was translated and the translation was evaluated through cognitive interviews. In the reliability-testing phase, the bilingual SSADDA was administered to 40 ethnic Samoans, including a sub-sample of 26 individuals who were retested. Cognitive interviews indicated the initial translation was culturally and linguistically appropriate except items pertaining to alcohol tolerance, which were modified to reflect Samoan concepts. SSADDA reliability testing indicated diagnoses of DSM-III-R and DSM-IV alcohol dependence were reliable. Reliability varied by language of administration. The English/Samoan version of the SSADDA is appropriate for the diagnosis of DSM-III-R alcohol dependence, which may be useful in advancing research and public health efforts to address alcohol problems in American Samoa and the Western Pacific. The translation methods may inform researchers translating diagnostic and assessment tools into different languages and cultures. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

Guidance for Subaqueous Dredged Material Capping.

DTIC Science & Technology

1998-06-01

from Ambrose Channel , over the contaminated sediments. At least two intermediate sur- veys and additional capping were required before capping was...organisms to a given bioturbation depth; reducing contami- nant flux rates to achieve specific sediment, pore water, or water column target...bathymetry, bottom slopes, cur- rents, water depths, water column density stratification, erosion/accretion trends, proximity to navigation channels

Sapovirus Translation Requires an Interaction between VPg and the Cap Binding Protein eIF4E

PubMed Central

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song

2014-01-01

viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation. PMID:25142584

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

PubMed

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song; Goodfellow, Ian; Cho, Kyoung-Oh

2014-11-01

functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation. Copyright © 2014 Hosmillo et al.

Mechanisms of Translation Control Underlying Long-lasting Synaptic Plasticity and the Consolidation of Long-term Memory

PubMed Central

Santini, Emanuela; Huynh, Thu N.; Klann, Eric

2018-01-01

The complexity of memory formation and its persistence is a phenomenon that has been studied intensely for centuries. Memory exists in many forms and is stored in various brain regions. Generally speaking, memories are reorganized into broadly distributed cortical networks over time through systems level consolidation. At the cellular level, storage of information is believed to initially occur via altered synaptic strength by processes such as long-term potentiation (LTP). New protein synthesis is required for long-lasting synaptic plasticity as well as for the formation of long-term memory. The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent protein synthesis and is required for numerous forms of long-lasting synaptic plasticity and long-term memory. As such, the study of mTORC1 and protein factors that control translation initiation and elongation have enhanced our understanding of how the process of protein synthesis is regulated during memory formation. Herein we will discuss the molecular mechanisms that regulate protein synthesis as well as pharmacological and genetic manipulations that demonstrate the requirement for proper translational control in long-lasting synaptic plasticity and long-term memory formation. PMID:24484700

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

PubMed Central

2012-01-01

Background Whole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells. Results The highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts. Conclusions BTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors. PMID:22559081

Porcine reproductive and respiratory syndrome virus infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff.

PubMed

Li, Yang; Fang, Liurong; Zhou, Yanrong; Tao, Ran; Wang, Dang; Xiao, Shaobo

2018-06-13

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest. IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation

Targeted in-vivo computed tomography (CT) imaging of tissue ACE using concentrated lisinopril-capped gold nanoparticle solutions

NASA Astrophysics Data System (ADS)

Daniel, Marie-Christine; Aras, Omer; Smith, Mark F.; Nan, Anjan; Fleiter, Thorsten

2010-04-01

The development of cardiac and pulmonary fibrosis have been associated with overexpression of angiotensin-converting enzyme (ACE). Moreover, ACE inhibitors, such as lisinopril, have shown a benificial effect for patients diagnosed with heart failure or systemic hypertension. Thus targeted imaging of the ACE is of crucial importance for monitoring of the tissue ACE activity as well as the treatment efficacy in heart failure. In this respect, lisinopril-capped gold nanoparticles were prepared to provide a new type of probe for targeted molecular imaging of ACE by tuned K-edge computed tomography (CT) imaging. Concentrated solutions of these modified gold nanoparticles, with a diameter around 16 nm, showed high contrast in CT imaging. These new targeted imaging agents were thus used for in vivo imaging on rat models.

Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

PubMed

Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

2018-06-26

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

The effect of polar caps on obliquity

NASA Technical Reports Server (NTRS)

Lindner, B. L.

1993-01-01

Rubincam has shown that the Martian obliquity is dependent on the seasonal polar caps. In particular, Rubincam analytically derived this dependence and showed that the change in obliquity is directly proportional to the seasonal polar cap mass. Rubincam concludes that seasonal friction does not appear to have changed Mars' climate significantly. Using a computer model for the evolution of the Martian atmosphere, Haberle et al. have made a convincing case for the possibility of huge polar caps, about 10 times the mass of the current polar caps, that exist for a significant fraction of the planet's history. Since Rubincam showed that the effect of seasonal friction on obliquity is directly proportional to polar cap mass, a scenario with a ten-fold increase in polar cap mass over a significant fraction of the planet's history would result in a secular increase in Mars' obliquity of perhaps 10 degrees. Hence, the Rubincam conclusion of an insignificant contribution to Mars' climate by seasonal friction may be incorrect. Furthermore, if seasonal friction is an important consideration in the obliquity of Mars, this would significantly alter the predictions of past obliquity.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis.

PubMed

Spangle, Jennifer M; Münger, Karl

2010-09-01

The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors

PubMed Central

Pflug, Alexander; Gaudon, Stephanie; Resa-Infante, Patricia; Lethier, Mathilde; Reich, Stefan; Schulze, Wiebke M

2018-01-01

Abstract Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation (’priming state’) and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the ‘apo’ state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the ‘apo’ state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive ‘apo’ state. PMID:29202182

Culture in Translation and Translation Studies.

ERIC Educational Resources Information Center

Schaffner, Christina

1994-01-01

Reviews the evolution of translation from an exchange of information within and across cultural boundaries to its current status as a scholarly endeavor. Translations may have far-reaching effects in the target and source culture. Translators should be cognizant of the foreign language and culture in order to successfully realize their role as…

Silibinin inhibits translation initiation: implications for anticancer therapy.

PubMed

Lin, Chen-Ju; Sukarieh, Rami; Pelletier, Jerry

2009-06-01

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Therapeutic Targeting of Alternative Translation Initiation in Breast Cancer

DTIC Science & Technology

2009-04-01

investigation within the next 6 months. Cell type specific cancer cell killing of the prototype oncolytic poliovirus , PVS-RIPO, depends on selective...demanded by FDA. 15. SUBJECT TERMS Translation, eIF4E, eIF4G, IRES, Cancer, Poliovirus 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...genetically recombinant poliovirus . Moreover, my work has laid the groundwork for correlative testing and efficacy studies of a vast array of protein kinase

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene

Translation Quality Assessment in Health Research: A Functionalist Alternative to Back-Translation.

PubMed

Colina, Sonia; Marrone, Nicole; Ingram, Maia; Sánchez, Daisey

2017-09-01

As international research studies become more commonplace, the importance of developing multilingual research instruments continues to increase and with it that of translated materials. It is therefore not unexpected that assessing the quality of translated materials (e.g., research instruments, questionnaires, etc.) has become essential to cross-cultural research, given that the reliability and validity of the research findings crucially depend on the translated instruments. In some fields (e.g., public health and medicine), the quality of translated instruments can also impact the effectiveness and success of interventions and public campaigns. Back-translation (BT) is a commonly used quality assessment tool in cross-cultural research. This quality assurance technique consists of (a) translation (target text [TT 1 ]) of the source text (ST), (b) translation (TT 2 ) of TT 1 back into the source language, and (c) comparison of TT 2 with ST to make sure there are no discrepancies. The accuracy of the BT with respect to the source is supposed to reflect equivalence/accuracy of the TT. This article shows how the use of BT as a translation quality assessment method can have a detrimental effect on a research study and proposes alternatives to BT. One alternative is illustrated on the basis of the translation and quality assessment methods used in a research study on hearing loss carried out in a border community in the southwest of the United States.

Pharmacogenetic Inhibition of eIF4E-Dependent Mmp9 mRNA Translation Reverses Fragile X Syndrome-like Phenotypes

PubMed Central

Gkogkas, Christos G.; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J.; Konicek, Bruce W.; Graff, Jeremy R.; Tzinia, Athina K.; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-01-01

SUMMARY Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS pheno-types. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. PMID:25466251

Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

PubMed

Gkogkas, Christos G; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J; Konicek, Bruce W; Graff, Jeremy R; Tzinia, Athina K; Lacaille, Jean-Claude; Sonenberg, Nahum

2014-12-11

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Translation invariant time-dependent massive gravity: Hamiltonian analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mourad, Jihad; Steer, Danièle A.; Noui, Karim, E-mail: mourad@apc.univ-paris7.fr, E-mail: karim.noui@lmpt.univ-tours.fr, E-mail: steer@apc.univ-paris7.fr

2014-09-01

The canonical structure of the massive gravity in the first order moving frame formalism is studied. We work in the simplified context of translation invariant fields, with mass terms given by general non-derivative interactions, invariant under the diagonal Lorentz group, depending on the moving frame as well as a fixed reference frame. We prove that the only mass terms which give 5 propagating degrees of freedom are the dRGT mass terms, namely those which are linear in the lapse. We also complete the Hamiltonian analysis with the dynamical evolution of the system.

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

PubMed Central

Bélanger, François; Stepinski, Janusz; Darzynkiewicz, Edward; Pelletier, Jerry

2010-01-01

Cellular eukaryotic mRNAs are capped at their 5′ ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2′-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2′-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo. PMID:20713356

Flavivirus RNA cap methyltransferase: structure, function, and inhibition.

PubMed

Liu, Lihui; Dong, Hongping; Chen, Hui; Zhang, Jing; Ling, Hua; Li, Zhong; Shi, Pei-Yong; Li, Hongmin

2010-08-01

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5' terminal cap 1 structure (m(7)GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m(7)GpppA → m(7)GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.

Hepatic translation control in the late-gestation fetal rat.

PubMed

Gruppuso, Philip A; Tsai, Shu-Whei; Boylan, Joan M; Sanders, Jennifer A

2008-08-01

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4A1 and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma.

PubMed

Holmes, Brent; Lee, Jihye; Landon, Kenna A; Benavides-Serrato, Angelica; Bashir, Tariq; Jung, Michael E; Lichtenstein, Alan; Gera, Joseph

2016-07-01

Our previous work has demonstrated an intrinsic mRNA-specific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

PubMed Central

Natsuizaka, Mitsuteru; Naganuma, Seiji; Kagawa, Shingo; Ohashi, Shinya; Ahmadi, Azal; Subramanian, Harry; Chang, Sanders; Nakagawa, Kei J.; Ji, Xinjun; Liebhaber, Stephen A.; Klein-Szanto, Andres J.; Nakagawa, Hiroshi

2012-01-01

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis. PMID:22415309

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

PubMed

Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2018-01-01

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

Targeting MUC1-C inhibits the AKT-S6K1-elF4A pathway regulating TIGAR translation in colorectal cancer.

PubMed

Ahmad, Rehan; Alam, Maroof; Hasegawa, Masanori; Uchida, Yasumitsu; Al-Obaid, Omar; Kharbanda, Surender; Kufe, Donald

2017-02-02

Colorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 heterodimeric protein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. Here, we investigate the effects of the MUC1-C subunit inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on human colorectal cancer cells. TIGAR mRNA level was determined using qRT-PCR. Western blotting was used to measure TIGAR protein level and AKT-mTOR-S6K1 pathways. Reactive oxygen species and apoptosis were measured by flow cytometry. Effect of MUC1-C peptide, GO-203 was studied on colorectal xenograft tumors. Immunohistochemistry was utilized for TIGAR staining. Treatment of MUC1-overexpressing SKCO-1 and Colo-205 colon cancer cells with GO-203 was associated with downregulation of the TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases in GSH levels. GO-203 treatment also resulted in increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and as xenografts in nude mice. Inhibition of MUC1-C also downregulated TIGAR expression in xenograft tissues. These findings indicate that MUC1-C is a potential target for the treatment of colorectal cancer. Colorectal cancer patients who overexpress MUC1-C may be candidates for treatment with the MUC1-C inhibitor alone or in combination therapy with other agents.

SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana.

PubMed

Castro, Pedro Humberto; Verde, Nuno; Lourenço, Tiago; Magalhães, Alexandre Papadopoulos; Tavares, Rui Manuel; Bejarano, Eduardo Rodríguez; Azevedo, Herlânder

2015-12-01

Post-translational modification mechanisms function as switches that mediate the balance between optimum growth and the response to environmental stimuli, by regulating the activity of key proteins. SUMO (small ubiquitin-like modifier) attachment, or sumoylation, is a post-translational modification that is essential for the plant stress response, also modulating hormonal circuits to co-ordinate developmental processes. The Arabidopsis SUMO E3 ligase SAP and Miz 1 (SIZ1) is the major SUMO conjugation enhancer in response to stress, and is implicated in several aspects of plant development. Here we report that known SUMO targets are over-represented in multiple carbohydrate-related proteins, suggesting a functional link between sumoylation and sugar metabolism and signaling in plants. We subsequently observed that SUMO-conjugated proteins accumulate in response to high doses of sugar in a SIZ1-dependent manner, and that the null siz1 mutant displays increased expression of sucrose and starch catabolic genes and shows reduced starch levels. We demonstrated that SIZ1 controls germination time and post-germination growth via osmotic and sugar-dependent signaling, respectively. Glucose was specifically linked to SUMO-sugar interplay, with high levels inducing root growth inhibition and aberrant root hair morphology in siz1. The use of sugar analogs and sugar marker gene expression analysis allowed us to implicate SIZ1 in a signaling pathway dependent on glucose metabolism, probably involving modulation of SNF1-related kinase 1 (SnRK1) activity. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A nucleolar targeting signal in PML-I addresses PML to nucleolar caps in stressed or senescent cells.

PubMed

Condemine, Wilfried; Takahashi, Yuki; Le Bras, Morgane; de Thé, Hugues

2007-09-15

The promyelocytic leukemia (PML) tumour suppressor is the organiser of PML nuclear bodies, which are domains the precise functions of which are still disputed. We show that upon several types of stress, endogenous PML proteins form nucleolar caps and eventually engulf nucleolar components. Only two specific PML splice variants (PML-I and PML-IV) are efficiently targeted to the nucleolus and the abundant PML-I isoform is required for the targeting of endogenous PML proteins to this organelle. We identified a nucleolar targeting domain within the evolutionarily conserved C-terminus of PML-I. This domain contains a predicted exonuclease III fold essential for the targeting of the PML-I C-terminus to nucleolar fibrillar centres. Furthermore, spontaneous or oncogene retrieval-induced senescence is associated with the formation of very large PML nuclear bodies that initially contain nucleolar components. Later, poly-ubiquitin conjugates are found on the outer shell or within most of these senescence-associated PML bodies. Thus, unexpectedly, the scarcely studied PML-I isoform links PML bodies, nucleolus, senescence and proteolysis.

Identification of small molecule inhibitors of the Chikungunya virus nsP1 RNA capping enzyme.

PubMed

Feibelman, Kristen M; Fuller, Benjamin P; Li, Linfeng; LaBarbera, Daniel V; Geiss, Brian J

2018-06-01

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies. Copyright © 2018. Published by Elsevier B.V.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Dose Comparisons for a Site-specific Representative Person Using the Age-dependent Dose Coefficients in CAP88-PC Version 4.

PubMed

Stagich, Brooke H; Moore, Kelsey R; Newton, Joseph R; Dixon, Kenneth L; Jannik, G Timothy

2017-04-01

Most U.S. Department of Energy (DOE) facilities with radiological airborne releases use the U.S. Environmental Protection Agency's (EPA) environmental dosimetry code CAP88-PC to demonstrate compliance with regulations in 40CFR61, subpart H [National Emission Standards for Hazardous Air Pollutants: Radiological (NESHAP)]. In 2015, EPA released Version 4 of CAP88-PC, which included significant modifications that improved usability and age-dependent dose coefficients and usage factors for six age groups (infant, 1 y, 5 y, 10 y, 15 y, and adult). However, EPA has not yet provided specific guidance on how to use these age-dependent factors. For demonstrating compliance with DOE public dose regulations, the Savannah River Site (SRS) recently changed from using the maximally exposed individual (MEI) concept (adult male) to the representative person concept (age- and gender-averaged reference person). In this study, dose comparisons are provided between the MEI and a SRS-specific representative person using the age-specific dose coefficients and usage factors in CAP88-PC V.4. Dose comparisons also are provided for each of the six age groups using five radionuclides of interest at SRS (tritium oxide, Cs, Sr, Pu, and I). In general, the total effective dose increases about 11% for the representative person as compared to the current NESHAP MEI because of the inclusion of the more radiosensitive age groups.

Cervical Cap

MedlinePlus

... Videos for Educators Search English Español The Cervical Cap KidsHealth / For Teens / The Cervical Cap What's in ... Call the Doctor? Print What Is a Cervical Cap? A cervical cap is a small cup made ...

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

PubMed Central

Kabachinski, Greg; Kielar-Grevstad, D. Michelle; Zhang, Xingmin; James, Declan J.; Martin, Thomas F. J.

2016-01-01

The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. PMID:26700319

Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

PubMed Central

Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

2002-01-01

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

Alkyl-Capped Silicon Nanocrystals Lack Cytotoxicity and have Enhanced Intracellular Accumulation in Malignant Cells via Cholesterol-Dependent Endocytosis

PubMed Central

Alsharif, Naif H; Berger, Christine E M; Varanasi, Satya S; Chao, Yimin; Horrocks, Benjamin R; Datta, Harish K

2009-01-01

Nanocrystals of various inorganic materials are being considered for application in the life sciences as fluorescent labels and for such therapeutic applications as drug delivery or targeted cell destruction. The potential applications of the nanoparticles are critically compromised due to the well-documented toxicity and lack of understanding about the mechanisms involved in the intracellular internalization. Here intracellular internalization and toxicity of alkyl-capped silicon nanocrystals in human neoplastic and normal primary cells is reported. The capped nanocrystals lack cytotoxicity, and there is a marked difference in the rate and extent of intracellular accumulation of the nanoparticles between human cancerous and non-cancerous primary cells, the rate and extent being higher in the malignant cells compared to normal human primary cells. The exposure of the cells to the alkyl-capped nanocrystals demonstrates no evidence of in vitro cytotoxicity when assessed by cell morphology, apoptosis, and cell viability assays. The internalization of the nanocrystals by Hela and SW1353 cells is almost completely blocked by the pinocytosis inhibitors filipin, cytochalasin B, and actinomycin D. The internalization process is not associated with any surface change in the nanoparticles, as their luminescence spectrum is unaltered upon transport into the cytosol. The observed dramatic difference in the rate and extent of internalization of the nanocrystals between malignant and non-malignant cells therefore offers potential application in the management of human neoplastic conditions. PMID:19058285

Dynamic m(6)A mRNA methylation directs translational control of heat shock response.

PubMed

Zhou, Jun; Wan, Ji; Gao, Xiangwei; Zhang, Xingqian; Jaffrey, Samie R; Qian, Shu-Bing

2015-10-22

The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

Translation Stress Positively Regulates MscL-Dependent Excretion of Cytoplasmic Proteins

PubMed Central

Morra, Rosa; Del Carratore, Francesco; Muhamadali, Howbeer; Horga, Luminita Gabriela; Halliwell, Samantha

2018-01-01

ABSTRACT The apparent mislocalization or excretion of cytoplasmic proteins is a commonly observed phenomenon in both bacteria and eukaryotes. However, reports on the mechanistic basis and the cellular function of this so-called “nonclassical protein secretion” are limited. Here we report that protein overexpression in recombinant cells and antibiotic-induced translation stress in wild-type Escherichia coli cells both lead to excretion of cytoplasmic protein (ECP). Condition-specific metabolomic and proteomic analyses, combined with genetic knockouts, indicate a role for both the large mechanosensitive channel (MscL) and the alternative ribosome rescue factor A (ArfA) in ECP. Collectively, the findings indicate that MscL-dependent protein excretion is positively regulated in response to both osmotic stress and arfA-mediated translational stress. PMID:29382730

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor–activated macrophages

PubMed Central

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-01-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages. PMID:22675026

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

PubMed

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-08-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

[Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE)].

PubMed

Master, Adam; Nauman, Alicja

2014-01-01

Translation initiation is a key rate-limiting step in cellular protein synthesis. A cap-dependent initiation is the most effective mechanism of the translation. However, some physiological (mitosis) and pathological (oxidative stress) processes may switch the classic mechanism to an alternative one that is regulated by an mRNA element such as IRES, uORF, IRE, CPE, DICE, AURE or CITE. A recently discovered mechanism of RNA hypoxia response element (rHRE)-dependent translation initiation, may change the view of oxygen-regulated translation and give a new insight into unexplained biochemical processes. Hypoxia is one of the better-known factors that may trigger an alternative mechanism of the translation initiation. Temporal events of oxygen deficiency within tissues and organs may activate processes such as angiogenesis, myogenesis, regeneration, wound healing, and may promote an adaptive response in cardiovascular and neurodegenerative diseases. On the other hand, growth of solid tumors may be accompanied by cyclic hypoxia, allowing for synthesis of proteins required for further progression of cancer cells. This paper provides a review of current knowledge on translational control in the context of alternative models of translation initiation.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Views of Translational Research from a Somewhat Translational Scientist

PubMed Central

Talman, William T.

2013-01-01

This review arose from a talk entitled “Identifying Targets” and given by the author at EB2011 at the invitation of the American Federation for Medical Research (AFMR). The presentation was part of the AFMR workshop entitled “Keys for Translation: Science and Strategy” and focused on identifying clinically relevant targets as a result of observations made during basic scientific studies. The review emphasizes that targets do not have to be the aim that drives basic discovery, but communication between the basic scientist and clinical investigators may aid recognition of such targets and their translation to clinical applications. Using one line of investigator-initiated research from his own laboratory as an example, the author emphasizes that basic discovery must be hypothesis driven and allowed to follow its logical sequence. Finding treatments, while always an aim of biomedical research, may arise as a result of basic studies that were not originally aimed at a target of translational research. PMID:22781556

Effect of capping layer on spin-orbit torques

NASA Astrophysics Data System (ADS)

Sun, Chi; Siu, Zhuo Bin; Tan, Seng Ghee; Yang, Hyunsoo; Jalil, Mansoor B. A.

2018-04-01

In order to enhance the magnitude of spin-orbit torque (SOT), considerable experimental works have been devoted to studying the thickness dependence of the different layers in multilayers consisting of heavy metal (HM), ferromagnet (FM), and capping layers. Here, we present a theoretical model based on the spin-drift-diffusion formalism to investigate the effect of the capping layer properties such as its thickness on the SOT observed in experiments. It is found that the spin Hall-induced SOT can be significantly enhanced by incorporating a capping layer with an opposite spin Hall angle to that of the HM layer. The spin Hall torque can be maximized by tuning the capping layer thickness. However, in the absence of the spin Hall effect (SHE) in the capping layer, the torque decreases monotonically with the capping layer thickness. Conversely, the spin Hall torque is found to decrease monotonically with the FM layer thickness, irrespective of the presence or absence of the SHE in the capping layer. All these trends are in correspondence with experimental observations. Finally, our model suggests that capping layers with a long spin diffusion length and high resistivity would also enhance the spin Hall torque.

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

PubMed

Hsu, Jessie Hao-Ru; Hubbell-Engler, Benjamin; Adelmant, Guillaume; Huang, Jialiang; Joyce, Cailin E; Vazquez, Francisca; Weir, Barbara A; Montgomery, Philip; Tsherniak, Aviad; Giacomelli, Andrew O; Perry, Jennifer A; Trowbridge, Jennifer; Fujiwara, Yuko; Cowley, Glenn S; Xie, Huafeng; Kim, Woojin; Novina, Carl D; Hahn, William C; Marto, Jarrod A; Orkin, Stuart H

2017-09-01

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR . ©2017 American Association for Cancer Research.

Sulfate-dependent Anaerobic Oxidation of Methane as a Generation Mechanism for Calcite Cap Rock in Gulf Coast Salt Domes

NASA Astrophysics Data System (ADS)

Caesar, K. H.; Kyle, R.; Lyons, T. W.; Loyd, S. J.

2015-12-01

Gulf Coast salt domes, specifically their calcite cap rocks, have been widely recognized for their association with significant reserves of crude oil and natural gas. However, the specific microbial reactions that facilitate the precipitation of these cap rocks are still largely unknown. Insight into the mineralization mechanism(s) can be obtained from the specific geochemical signatures recorded in these structures. Gulf Coast cap rocks contain carbonate and sulfur minerals that exhibit variable carbon (d13C) and sulfur isotope (δ34S) signatures. Calcite d13C values are isotopically depleted and show a large range of values from -1 to -52‰, reflecting a mixture of various carbon sources including a substantial methane component. These depleted carbon isotope compositions combined with the presence of abundant sulfide minerals in cap rocks have led to interpretations that invoke microbial sulfate reduction as an important carbonate mineral-yielding process in salt dome environments. Sulfur isotope data from carbonate-associated sulfate (CAS: trace sulfate incorporated within the carbonate mineral crystal lattice) provide a more direct proxy for aqueous sulfate in salt dome systems and may provide a means to directly fingerprint ancient sulfate reduction. We find CAS sulfur isotope compositions (δ34SCAS) significantly greater than those of the precursor Jurassic sulfate-salt deposits (which exhibit δ34S values of ~ +15‰). This implies that cap rock carbonate generation occurred via microbial sulfate reduction under closed-system conditions. The co-occurrence of depleted carbonate d13C values (< ~30‰) and the enriched δ34SCAS values are evidence for sulfate-dependent anaerobic oxidation of methane (AOM). AOM, which has been shown to yield extensive seafloor carbonate authigenesis, is also potentially partly responsible for the carbonate minerals of the Gulf Coast calcite cap rocks through concomitant production of alkalinity. Collectively, these data shed

Bean Soup Translation: Flexible, Linguistically-Motivated Syntax for Machine Translation

ERIC Educational Resources Information Center

Mehay, Dennis Nolan

2012-01-01

Machine translation (MT) systems attempt to translate texts from one language into another by translating words from a "source language" and rearranging them into fluent utterances in a "target language." When the two languages organize concepts in very different ways, knowledge of their general sentence structure, or…

Capping agents in nanoparticle synthesis: Surfactant and solvent system

NASA Astrophysics Data System (ADS)

Gulati, Shivani; Sachdeva, M.; Bhasin, K. K.

2018-05-01

The preparation of nanomaterials by organometallic precursors require a capping agent, which primarily acts as stabilizing agent and provide colloidal stability along with preventing agglomeration and stopping uncontrolled growth. Final morphology of nanocrystal largely depends on the type of capping agent which is adsorbed on the surface of nanocrystal. Thus capping agents are the keys to obtain the small-sized nanoparticles and are very frequently used in colloidal synthesis of nanoparticles to avoid its overgrowth.

PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

PubMed Central

Gao, Guozhen; Dhar, Surbhi

2017-01-01

Abstract The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator. PMID:28115626

A novel route for preparing 5′ cap mimics and capped RNAs: phosphate-modified cap analogues obtained via click chemistry† †Electronic supplementary information (ESI) available: Tables S1–S6 and Fig. S1–S10, experimental procedures, HPLC profiles, NMR and HRMS spectra. See DOI: 10.1039/c6sc02437h Click here for additional data file. Click here for additional data file.

PubMed Central

Walczak, Sylwia; Nowicka, Anna; Kubacka, Dorota; Fac, Kaja; Wanat, Przemyslaw; Mroczek, Seweryn; Kowalska, Joanna

2017-01-01

The significant biological role of the mRNA 5′ cap in translation initiation makes it an interesting subject for chemical modifications aimed at producing useful tools for the selective modulation of intercellular processes and development of novel therapeutic interventions. However, traditional approaches to the chemical synthesis of cap analogues are time-consuming and labour-intensive, which impedes the development of novel compounds and their applications. Here, we explore a different approach for synthesizing 5′ cap mimics, making use of click chemistry (CuAAC) to combine two mononucleotide units and yield a novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain. As a result, we synthesized a library of 36 mRNA cap analogues differing in the location of the triazole ring, the polyphosphate chain length, and the type of linkers joining the phosphate and the triazole moieties. After biochemical evaluation, we identified two analogues that, when incorporated into mRNA, produced transcripts translated with efficiency similar to compounds unmodified in the oligophosphate bridge obtained by traditional synthesis. Moreover, we demonstrated that the triazole-modified cap structures can be generated at the RNA 5′ end using two alternative capping strategies: either the typical co-transcriptional approach, or a new post-transcriptional approach based on CuAAC. Our findings open new possibilities for developing chemically modified mRNAs for research and therapeutic applications, including RNA-based vaccinations. PMID:28451173

Preparation of near-infrared-labeled targeted contrast agents for clinical translation

NASA Astrophysics Data System (ADS)

Olive, D. Michael

2011-03-01

Targeted fluorophore-labeled contrast agents are moving toward translation to human surgical use. To prepare for future clinical use, we examined the performance of potential ligands targeting the epidermal growth factor receptor, α5β3 integrins, and GLUT transporters for their suitability as directed contrast agents. Each agent was labeled with IRDye 800CW, and near-infrared dye with excitation/emission wavelengths of 789/805 nm, which we determined had favorable toxicity characteristics. The probe molecules examined consisted of Affibodies, nanobodies, peptides, and the sugar 2-deoxy-D-glucose. Each probe was tested for specific and non-specific binding in cell based assays. All probe types showed good performance in mouse models for detecting either spontaneous tumors or tumor xenografts in vivo. Each of the probes tested show promise for future human clinical studies.

cap alpha. -Methylglucoside satisfies only Na/sup +/-dependent transport system of intestinal epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimmich, G.A.; Randles, J.

1981-01-01

The unidirectional influx of ..cap alpha..-methylglucoside (..cap alpha..-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na/sup +/, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance ..cap alpha..-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar canmore » be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.« less

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Autism-related deficits via dysregulated eIF4E-dependent translational control.

PubMed

Gkogkas, Christos G; Khoutorsky, Arkady; Ran, Israeli; Rampakakis, Emmanouil; Nevarko, Tatiana; Weatherill, Daniel B; Vasuta, Cristina; Yee, Stephanie; Truitt, Morgan; Dallaire, Paul; Major, François; Lasko, Paul; Ruggero, Davide; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2013-01-17

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma.

PubMed

Venkatesh, Humsa S; Tam, Lydia T; Woo, Pamelyn J; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M; Ni, Jing; Duveau, Damien Y; Morris, Patrick J; Zhao, Jean J; Thomas, Craig J; Monje, Michelle

2017-09-28

High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

PubMed Central

Venkatesh, Humsa S.; Tam, Lydia T.; Woo, Pamelyn J.; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M.; Ni, Jing; Duveau, Damien Y.; Morris, Patrick J.; Zhao, Jean J.; Thomas, Craig J.; Monje, Michelle

2017-01-01

Summary High-grade gliomas (HGG) are a devastating group of cancers, representing the leading cause of brain tumor-related death in both children and adults. Therapies aimed at mechanisms intrinsic to the glioma cell have translated to only limited success; effective therapeutic strategies will need to also target elements of the tumor microenvironment that promote glioma progression. We recently demonstrated that neuronal activity robustly promotes the growth of a range of molecularly and clinically distinct HGG types, including adult glioblastoma (GBM), anaplastic oligodendroglioma, pediatric GBM, and diffuse intrinsic pontine glioma (DIPG)1. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway1. However, neuroligin-3 necessity to glioma growth, proteolytic mechanism of secretion and further molecular consequences in glioma remain to be clarified. Here, we demonstrate a striking dependence of HGG growth on microenvironmental neuroligin-3, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy. PMID:28959975

47 CFR 61.48 - Transition rules for price cap formula calculations.

Code of Federal Regulations, 2014 CFR

2014-10-01

... percentage of Base Period Price Cap Revenues at the holding company level greater than the industry wide... filing entity is shifted to the CMT basket within price caps. Pooled local switching revenue will not be... section, to pooled elements after the Average Traffic Sensitive Charge reaches the target level. For the...

47 CFR 61.48 - Transition rules for price cap formula calculations.

Code of Federal Regulations, 2012 CFR

2012-10-01

... percentage of Base Period Price Cap Revenues at the holding company level greater than the industry wide... filing entity is shifted to the CMT basket within price caps. Pooled local switching revenue will not be... section, to pooled elements after the Average Traffic Sensitive Charge reaches the target level. For the...

47 CFR 61.48 - Transition rules for price cap formula calculations.

Code of Federal Regulations, 2013 CFR

2013-10-01

... percentage of Base Period Price Cap Revenues at the holding company level greater than the industry wide... filing entity is shifted to the CMT basket within price caps. Pooled local switching revenue will not be... section, to pooled elements after the Average Traffic Sensitive Charge reaches the target level. For the...

Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells

PubMed Central

Tang, Xun; Zhang, Xiao; Qiao, Yongxia; Shi, Yuling; Xu, Yanfeng; Wang, Zhongyong; Yu, Yongchun; Sun, Fenyong

2015-01-01

Doxorubicin (Doxo) is one of the most widely used chemotherapeutic drugs for patients with hepatocellular carcinoma (HCC). Doxo is a DNA intercalating drug that inhibits topoisomerase II. Thereby Doxo has the ability to block DNA replication and induce apoptosis. However, the other targets and mechanisms through which Doxo induces apoptosis to treat HCC still remain unknown. Here, we identified Mucosal vascular addressin cell adhesion molecule 1 (Madcam1) as a potential Doxo target because Madcam1 overexpression suppressed, while Madcam1 depletion stimulated Doxo-induced apoptosis. Furthermore, we first revealed that Doxo can induce apoptosis by blocking protein translation initiation. In contrast, Madcam1 activated protein translation through an opposite mechanism. We also found de-phosphorylation of AKT may be an important pro-apoptotic event that is triggered by Doxo-induced Madcam1 down-regulation. Finally, we revealed that Madcam1 promoted increased AKT phosphorylation, which is essential for maintaining the sensitivity of HCC cells to Doxo treatment. Taken together, we uncovered a potential mechanism for Doxo-induced apoptosis in HCC treatment through targeting Madcam1 and AKT and blocking protein translation initiation. PMID:26124182

Mdm2 mediates FMRP- and Gp1 mGluR-dependent protein translation and neural network activity.

PubMed

Liu, Dai-Chi; Seimetz, Joseph; Lee, Kwan Young; Kalsotra, Auinash; Chung, Hee Jung; Lu, Hua; Tsai, Nien-Pei

2017-10-15

Activating Group 1 (Gp1) metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, elicits translation-dependent neural plasticity mechanisms that are crucial to animal behavior and circuit development. Dysregulated Gp1 mGluR signaling has been observed in numerous neurological and psychiatric disorders. However, the molecular pathways underlying Gp1 mGluR-dependent plasticity mechanisms are complex and have been elusive. In this study, we identified a novel mechanism through which Gp1 mGluR mediates protein translation and neural plasticity. Using a multi-electrode array (MEA) recording system, we showed that activating Gp1 mGluR elevates neural network activity, as demonstrated by increased spontaneous spike frequency and burst activity. Importantly, we validated that elevating neural network activity requires protein translation and is dependent on fragile X mental retardation protein (FMRP), the protein that is deficient in the most common inherited form of mental retardation and autism, fragile X syndrome (FXS). In an effort to determine the mechanism by which FMRP mediates protein translation and neural network activity, we demonstrated that a ubiquitin E3 ligase, murine double minute-2 (Mdm2), is required for Gp1 mGluR-induced translation and neural network activity. Our data showed that Mdm2 acts as a translation suppressor, and FMRP is required for its ubiquitination and down-regulation upon Gp1 mGluR activation. These data revealed a novel mechanism by which Gp1 mGluR and FMRP mediate protein translation and neural network activity, potentially through de-repressing Mdm2. Our results also introduce an alternative way for understanding altered protein translation and brain circuit excitability associated with Gp1 mGluR in neurological diseases such as FXS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

PubMed

Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

2015-04-29

One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

Time-Resolved Intrafraction Target Translations and Rotations During Stereotactic Liver Radiation Therapy: Implications for Marker-based Localization Accuracy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertholet, Jenny, E-mail: jennbe@rm.dk; Worm, Esben S.; Fledelius, Walther

Purpose: Image guided liver stereotactic body radiation therapy (SBRT) often relies on implanted fiducial markers. The target localization accuracy decreases with increased marker-target distance. This may occur partly because of liver rotations. The aim of this study was to examine time-resolved translations and rotations of liver marker constellations and investigate if time-resolved intrafraction rotational corrections can improve localization accuracy in liver SBRT. Methods and Materials: Twenty-nine patients with 3 implanted markers received SBRT in 3 to 6 fractions. The time-resolved trajectory of each marker was estimated from the projections of 1 to 3 daily cone beam computed tomography scans andmore » used to calculate the translation and rotation of the marker constellation. In all cone beam computed tomography projections, the time-resolved position of each marker was predicted from the position of another surrogate marker by assuming that the marker underwent either (1) the same translation as the surrogate marker; or (2) the same translation as the surrogate marker corrected by the rotation of the marker constellation. The localization accuracy was quantified as the root-mean-square error (RMSE) between the estimated and the actual marker position. For comparison, the RMSE was also calculated when the marker's position was estimated as its mean position for all the projections. Results: The mean translational and rotational range (2nd-98th percentile) was 2.0 mm/3.9° (right-left), 9.2 mm/2.9° (superior-inferior), 4.0 mm/4.0° (anterior-posterior), and 10.5 mm (3-dimensional). Rotational corrections decreased the mean 3-dimensional RMSE from 0.86 mm to 0.54 mm (P<.001) and halved the RMSE increase per millimeter increase in marker distance. Conclusions: Intrafraction rotations during liver SBRT reduce the accuracy of marker-guided target localization. Rotational correction can improve the localization accuracy with a factor of approximately 2

The mechanism of RNA 5' capping with NAD +, NADH and desphospho-CoA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bird, Jeremy G.; Zhang, Yu; Tian, Yuan

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, inmore » a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.« less

Differential expression of mucins 1-6 in papillary thyroid carcinoma: evidence for transformation-dependent post-translational modifications of MUC1 in situ.

PubMed

Magro, Gaetano; Schiappacassi, Monica; Perissinotto, Daniela; Corsaro, Antonella; Borghese, Cinzia; Belfiore, Antonino; Colombatti, Alfonso; Grasso, Sebastiano; Botti, Carlo; Bombardieri, Emilio; Perris, Roberto

2003-07-01

Mucins are primary glycoproteins of epithelia that are known to undergo major changes in their post-translational processing during neoplastic transformation. This study has examined the expression pattern of seven primary mucins, ie mucin (MUC) 1, 2, 3, 4, 5AC, 5B and 6, in normal, hyperplastic, benign neoplastic, and papillary-type carcinoma (PTC) tissues of the thyroid. MUC1 and MUC5B were the only mucins to be widely transcribed in both benign and malignant tissues. In contrast, MUC4 transcripts were undetectable in normal thyroids, and were present in only 40% of the hyperplastic and malignant thyroid tissues. In PTC, MUC1 was identified as a single mRNA transcript, rejecting the idea that this mucin may undergo transformation-dependent alternative splicing in thyroid tumours. The tissue distribution of MUC1 and MUC4 proteins was highly heterogeneous: this largely paralleled their mRNA expression profiles and supported the conclusion that whereas MUC1 was ubiquitously expressed in PTC, MUC4 was detectable in less than 20% of the cases analysed. In order to determine whether post-translational modifications of MUC1, putatively associated with malignancy, also occurred in the mucin produced by PTC, immunohistochemistry was performed with a panel of well-characterized anti-MUC1 antibodies in conjunction with digestion of the tissue sections with deglycosylating enzymes. These experiments, which were supported by immunochemical analyses of the MUC1 and MUC4 glycoforms extracted from tissues, collectively demonstrated markedly divergent MUC1 glycosylation profiles in normal and benign thyroid tissues when compared with PTC. Characteristically, these latter neoplastic cells produced mucin molecules carrying complex poly-N-lactosamine-type glycans capped with fucose and neuraminic acid residues. The present study also found evidence in PTC for the potential presence of proteolytically processed MUC1 isoforms which differ in their post-translational traits depending

Feasibility of 3D printed air slab diode caps for small field dosimetry.

PubMed

Perrett, Benjamin; Charles, Paul; Markwell, Tim; Kairn, Tanya; Crowe, Scott

2017-09-01

Commercial diode detectors used for small field dosimetry introduce a field-size-dependent over-response relative to an ideal, water-equivalent dosimeter due to high density components in the body of the detector. An air gap above the detector introduces a field-size-dependent under-response, and can be used to offset the field-size-dependent detector over-response. Other groups have reported experimental validation of caps containing air gaps for use with several types of diodes in small fields. This paper examines two designs for 3D printed diode air caps for the stereotactic field diode (SFD)-a cap containing a sealed air cavity, and a cap with an air cavity at the face of the SFD. Monte Carlo simulations of both designs were performed to determine dimensions for an air cavity to introduce the desired dosimetric correction. Various parameter changes were also simulated to estimate the dosimetric uncertainties introduced by 3D printing. Cap layer dimensions, cap density changes due to 3D printing, and unwanted air gaps were considered. For the sealed design the optimal air gap size for water-equivalent cap material was 0.6 mm, which increased to 1.0 mm when acrylonitrile butadiene styrene in the cap was simulated. The unsealed design had less variation, a 0.4 mm air gap is optimal in both situations. Unwanted air pockets in the bore of the cap and density changes introduced by the 3D printing process can potentially introduce significant dosimetric effects. These effects may be limited by using fine print resolutions and minimising the volume of cap material.

Bronchoalveolar carcinoma (adenocarcinoma) mimicking recurrent bacterial community-acquired pneumonia (CAP).

PubMed

Cunha, Burke A; Syed, Uzma; Mikail, Nardeen

2012-01-01

Depending on the community-acquired pneumonia (CAP) pathogen, host factors, and immune status, CAPs resolve on chest x-rays at different rates. CAPs that resolve more slowly than expected, or not at all, are termed "slowly or non-resolving CAPs." In contrast, recurrent CAPs may be due to host defense defects (eg, multiple myelomas) or post-obstructive bronchogenic carcinomas. There are a variety of noninfectious disorders that may mimic CAPs on chest x-ray: alveolar hemorrhage, pulmonary drug reactions, radiation pneumonitis, Wegener's granulomatosis, bronchiolitis obliterans organizing pneumonia, bronchogenic carcinomas, and lymphomas. Noninfectious mimics of recurrent CAPs include congestive heart failure, pulmonary emboli, infarctions, sarcoidosis, and systemic lupus erythematosus pneumonitis. We present the case of a middle-aged man who presented with recurrent right middle lobe and right lower lobe CAPs. Diagnostic bronchoscopy showed no bronchial obstruction, but open lung biopsy showed bronchoalveolar carcinoma (well-differentiated adenocarcinoma). Bronchoalveolar carcinomas presenting as post-obstructive or recurrent CAPs are rare because the spread is along tissue planes and not endobronchially. The case described demonstrates a rare cause of bronchogenic carcinoma mimicking recurrent CAP. Copyright © 2012 Elsevier Inc. All rights reserved.

Translating Proper Nouns: A Case Study on English Translation of Hafez's Lyrics

ERIC Educational Resources Information Center

Shirinzadeh, Seyed Alireza; Mahadi, Tengku Sepora Tengku

2014-01-01

Proper nouns are regarded so simple that they might be taken for granted in translation explorations. Some may believe that they should not be translated in transmitting source texts to target texts. But, it is not the case; if one looks at present translations, he will notice that different strategies might be applied for translating proper…

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

PubMed Central

Diaz, Arturo; Gallei, Andreas

2012-01-01

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell. PMID:22090102

Cradle Cap

MedlinePlus

Cradle cap Overview Cradle cap causes crusty or oily scaly patches on a baby's scalp. The condition isn't painful or itchy. But it can ... scales that aren't easy to remove. Cradle cap usually clears up on its own in a ...

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

PubMed

Kabachinski, Greg; Kielar-Grevstad, D Michelle; Zhang, Xingmin; James, Declan J; Martin, Thomas F J

2016-02-15

The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. © 2016 Kabachinski, Kielar-Grevstad, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Upper Thermosphere Winds and Temperatures in the Geomagnetic Polar Cap: Solar Cycle, Geomagnetic Activity, and Interplanetary Magnetic Field Dependencies

NASA Technical Reports Server (NTRS)

Killeen, T. L.; Won, Y.-I.; Niciejewski, R. J.; Burns, A. G.

1995-01-01

Ground-based Fabry-Perot interferometers located at Thule, Greenland (76.5 deg. N, 69.0 deg. W, lambda = 86 deg.) and at Sondre Stromfjord, Greenland (67.0 deg. N, 50.9 deg. W, lambda = 74 deg.) have monitored the upper thermospheric (approx. 240-km altitude) neutral wind and temperature over the northern hemisphere geomagnetic polar cap since 1983 and 1985, respectively. The thermospheric observations are obtained by determining the Doppler characteristics of the (OI) 15,867-K (630.0-nm) emission of atomic oxygen. The instruments operate on a routine, automatic, (mostly) untended basis during the winter observing seasons, with data coverage limited only by cloud cover and (occasional) instrument failures. This unique database of geomagnetic polar cap measurements now extends over the complete range of solar activity. We present an analysis of the measurements made between 1985 (near solar minimum) and 1991 (near solar maximum), as part of a long-term study of geomagnetic polar cap thermospheric climatology. The measurements from a total of 902 nights of observations are compared with the predictions of two semiempirical models: the Vector Spherical Harmonic (VSH) model of Killeen et al. (1987) and the Horizontal Wind Model (HWM) of Hedin et al. (1991). The results are also analyzed using calculations of thermospheric momentum forcing terms from the Thermosphere-ionosphere General Circulation Model TGCM) of the National Center for Atmospheric Research (NCAR). The experimental results show that upper thermospheric winds in the geomagnetic polar cap have a fundamental diurnal character, with typical wind speeds of about 200 m/s at solar minimum, rising to up to about 800 m/s at solar maximum, depending on geomagnetic activity level. These winds generally blow in the antisunward direction, but are interrupted by episodes of modified wind velocity and altered direction often associated with changes in the orientation of the Interplanetary Magnetic Field (IMF). The

Heating in short-pulse laser-driven cone-capped wire targets

NASA Astrophysics Data System (ADS)

Mason, R. J.; Wei, M.; King, J.; Beg, F.; Stephens, R. B.

2007-11-01

The 2-D implicit hybrid simulation code e-PLAS has been used to study heating in cone-capped copper wire targets. The code e-PLAS tracks collisional particle-in-cell (PIC) electrons traversing background plasma of collisional Eulerian cold electron and ion fluids. It computes E- and B-fields by the Implicit Moment Method [1,2]. In recent experiments [3] at the Vulcan laser facility, sub- picosecond laser pulses at 1.06 μm, and 4.0 x 10^20 W/cm^2 intensity were focused into thin-walled (˜10 μm) cones attached to copper wires. The wire diameter was varied from 10-40 μm with a typical length of 1 mm. We characterize heating of the wires as a function of their diameters and length, and relate modifications of this heating to changes in the assumed laser-generated hot electron spectrum and directivity. As in recent nail experiments [4], the cones can serve as reservoirs for hot electrons, diverting them from passage down the wires. [1] R. J. Mason, and C. Cranfill, IEEE Trans. Plasma Sci. PS-14, 45 (1986). [2] R. J. Mason, J. Comp. Phys. 71, 429 (1987). [3] J. King et al., to be submitted to Phys. Rev. Lett.. [4] R. J. Mason, M. Wei, F. Beg, R. Stephens, and C. Snell, in Proc. of ICOPS07, Albuquerque, NM, June 17-22, 2007, Talk 7D4.

The cervical cap.

PubMed

1988-10-07

The US Food and Drug Administration has approved marketing of the Prentif cavity-rim cervical cap. This contraceptive device is being distributed in the US and Canada by Cervical Cap Ltd, Los Gatos, California. The Prentif cap is available in 4 sizes: 22, 25, 28, and 31 mm inside diameter, with a length of 1 1/4-1 1/2 inches. In a multicenter trial involving 522 diaphragm users and 581 cap users followed for 2 years, the cap was 82.6% effective and the diaphragm was 83.3% effective in preventing pregnancy. When pregnancies attributable to user failure were excluded, these rates were increased to 93.6% for the cap and 95.4% for the diaphragm. 4% of cap users compared with only 1.7% of diaphragm users in this study developed abnormal Pap smears after 3 months of use; in addition, a higher proportion of cap users became infected with Gardnerella vaginalis and Monilia. Theoretical hazards include toxic shock syndrome and endometriosis due to backflow of menstrual fluids. Cap users are advised to undergo a Pap test after 3 months of use and discontinue cap use if the results are abnormal. The cap should not be used during menstruation. Although the cap can be left in place for up to 48 hours, its position should be checked before and after each episode of intercourse. The cervical cap requires less spermicide than the diaphragm and is not as messy. In addition, it can be left in the vagina twice as long as the diaphragm, without additional spermicide. Since the cap is smaller than the diaphragm and does not cover the vaginal wall, some women find intercourse more pleasurable with this device.

mTORC1-Mediated Inhibition of 4EBP1 Is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma.

PubMed

Wu, Chang-Chih; Hou, Shirui; Orr, Brent A; Kuo, Bryan R; Youn, Yong Ha; Ong, Taren; Roth, Fanny; Eberhart, Charles G; Robinson, Giles W; Solecki, David J; Taketo, Makoto M; Gilbertson, Richard J; Roussel, Martine F; Han, Young-Goo

2017-12-18

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Rolling Circle Translation of Circular RNA in Living Human Cells.

PubMed

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-11-10

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Rolling Circle Translation of Circular RNA in Living Human Cells

PubMed Central

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-01-01

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought. PMID:26553571

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

CAPS and Munc13: CATCHRs that SNARE Vesicles.

PubMed

James, Declan J; Martin, Thomas F J

2013-12-04

CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

PubMed

Ogino, Minako; Ogino, Tomoaki

2017-03-15

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

PubMed

Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

2016-07-15

In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems.

PubMed

Lam, Maggie P Y; Venkatraman, Vidya; Xing, Yi; Lau, Edward; Cao, Quan; Ng, Dominic C M; Su, Andrew I; Ge, Junbo; Van Eyk, Jennifer E; Ping, Peipei

2016-11-04

Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

Translational research: precision medicine, personalized medicine, targeted therapies: marketing or science?

PubMed

Marquet, Pierre; Longeray, Pierre-Henry; Barlesi, Fabrice; Ameye, Véronique; Augé, Pascale; Cazeneuve, Béatrice; Chatelut, Etienne; Diaz, Isabelle; Diviné, Marine; Froguel, Philippe; Goni, Sylvia; Gueyffier, François; Hoog-Labouret, Natalie; Mourah, Samia; Morin-Surroca, Michèle; Perche, Olivier; Perin-Dureau, Florent; Pigeon, Martine; Tisseau, Anne; Verstuyft, Céline

2015-01-01

Personalized medicine is based on: 1) improved clinical or non-clinical methods (including biomarkers) for a more discriminating and precise diagnosis of diseases; 2) targeted therapies of the choice or the best drug for each patient among those available; 3) dose adjustment methods to optimize the benefit-risk ratio of the drugs chosen; 4) biomarkers of efficacy, toxicity, treatment discontinuation, relapse, etc. Unfortunately, it is still too often a theoretical concept because of the lack of convenient diagnostic methods or treatments, particularly of drugs corresponding to each subtype of pathology, hence to each patient. Stratified medicine is a component of personalized medicine employing biomarkers and companion diagnostics to target the patients likely to present the best benefit-risk balance for a given active compound. The concept of targeted therapy, mostly used in cancer treatment, relies on the existence of a defined molecular target, involved or not in the pathological process, and/or on the existence of a biomarker able to identify the target population, which should logically be small as compared to the population presenting the disease considered. Targeted therapies and biomarkers represent important stakes for the pharmaceutical industry, in terms of market access, of return on investment and of image among the prescribers. At the same time, they probably represent only the first generation of products resulting from the combination of clinical, pathophysiological and molecular research, i.e. of translational research. © 2015 Société Française de Pharmacologie et de Thérapeutique.

Influence of thiol capping on the photoluminescence properties of L-cysteine-, mercaptoethanol- and mercaptopropionic acid-capped ZnS nanoparticles.

PubMed

Tiwari, A; Dhoble, S J; Kher, R S

2015-11-01

Mercaptoethanol (ME), mercaptopropionic acid (MPA) and L-cysteine (L-Cys) having -SH functional groups were used as surface passivating agents for the wet chemical synthesis of ZnS nanoparticles. The effect of the thiol group on the optical and photoluminescence (PL) properties of ZnS nanoparticles was studied. L-Cysteine-capped ZnS nanoparticles showed the highest PL intensity among the studied capping agents, with a PL emission peak at 455 nm. The PL intensity was found to be dependent on the concentration of Zn(2+) and S(2-) precursors. The effect of buffer on the PL intensity of L-Cys-capped ZnS nanoparticles was also studied. UV/Vis spectra showed blue shifting of the absorption edge. Copyright © 2015 John Wiley & Sons, Ltd.

Basic/Translational Development of Forthcoming Opioid- and Nonopioid-Targeted Pain Therapeutics.

PubMed

Knezevic, Nebojsa Nick; Yekkirala, Ajay; Yaksh, Tony L

2017-11-01

Opioids represent an efficacious therapeutic modality for some, but not all pain states. Singular reliance on opioid therapy for pain management has limitations, and abuse potential has deleterious consequences for patient and society. Our understanding of pain biology has yielded insights and opportunities for alternatives to conventional opioid agonists. The aim is to have efficacious therapies, with acceptable side effect profiles and minimal abuse potential, which is to say an absence of reinforcing activity in the absence of a pain state. The present work provides a nonexclusive overview of current drug targets and potential future directions of research and development. We discuss channel activators and blockers, including sodium channel blockers, potassium channel activators, and calcium channel blockers; glutamate receptor-targeted agents, including N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and metabotropic receptors. Furthermore, we discuss therapeutics targeted at γ-aminobutyric acid, α2-adrenergic, and opioid receptors. We also considered antagonists of angiotensin 2 and Toll receptors and agonists/antagonists of adenosine, purine receptors, and cannabinoids. Novel targets considered are those focusing on lipid mediators and anti-inflammatory cytokines. Of interest is development of novel targeting strategies, which produce long-term alterations in pain signaling, including viral transfection and toxins. We consider issues in the development of druggable molecules, including preclinical screening. While there are examples of successful translation, mechanistically promising preclinical candidates may unexpectedly fail during clinical trials because the preclinical models may not recapitulate the particular human pain condition being addressed. Molecular target characterization can diminish the disconnect between preclinical and humans' targets, which should assist in developing nonaddictive analgesics.

Fertility and polarized cell growth depends on eIF5A for translation of polyproline-rich formins in Saccharomyces cerevisiae.

PubMed

Li, Tianlu; Belda-Palazón, Borja; Ferrando, Alejandro; Alepuz, Paula

2014-08-01

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes. Copyright © 2014 by the Genetics Society of America.

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

PubMed Central

Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

2015-01-01

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

PubMed

Hoffman, Brett; Li, Zhubing; Liu, Qiang

2015-08-01

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

Knowledge translation of research findings

PubMed Central

2012-01-01

Background One of the most consistent findings from clinical and health services research is the failure to translate research into practice and policy. As a result of these evidence-practice and policy gaps, patients fail to benefit optimally from advances in healthcare and are exposed to unnecessary risks of iatrogenic harms, and healthcare systems are exposed to unnecessary expenditure resulting in significant opportunity costs. Over the last decade, there has been increasing international policy and research attention on how to reduce the evidence-practice and policy gap. In this paper, we summarise the current concepts and evidence to guide knowledge translation activities, defined as T2 research (the translation of new clinical knowledge into improved health). We structure the article around five key questions: what should be transferred; to whom should research knowledge be transferred; by whom should research knowledge be transferred; how should research knowledge be transferred; and, with what effect should research knowledge be transferred? Discussion We suggest that the basic unit of knowledge translation should usually be up-to-date systematic reviews or other syntheses of research findings. Knowledge translators need to identify the key messages for different target audiences and to fashion these in language and knowledge translation products that are easily assimilated by different audiences. The relative importance of knowledge translation to different target audiences will vary by the type of research and appropriate endpoints of knowledge translation may vary across different stakeholder groups. There are a large number of planned knowledge translation models, derived from different disciplinary, contextual (i.e., setting), and target audience viewpoints. Most of these suggest that planned knowledge translation for healthcare professionals and consumers is more likely to be successful if the choice of knowledge translation strategy is informed by

Knowledge translation of research findings.

PubMed

Grimshaw, Jeremy M; Eccles, Martin P; Lavis, John N; Hill, Sophie J; Squires, Janet E

2012-05-31

One of the most consistent findings from clinical and health services research is the failure to translate research into practice and policy. As a result of these evidence-practice and policy gaps, patients fail to benefit optimally from advances in healthcare and are exposed to unnecessary risks of iatrogenic harms, and healthcare systems are exposed to unnecessary expenditure resulting in significant opportunity costs. Over the last decade, there has been increasing international policy and research attention on how to reduce the evidence-practice and policy gap. In this paper, we summarise the current concepts and evidence to guide knowledge translation activities, defined as T2 research (the translation of new clinical knowledge into improved health). We structure the article around five key questions: what should be transferred; to whom should research knowledge be transferred; by whom should research knowledge be transferred; how should research knowledge be transferred; and, with what effect should research knowledge be transferred? We suggest that the basic unit of knowledge translation should usually be up-to-date systematic reviews or other syntheses of research findings. Knowledge translators need to identify the key messages for different target audiences and to fashion these in language and knowledge translation products that are easily assimilated by different audiences. The relative importance of knowledge translation to different target audiences will vary by the type of research and appropriate endpoints of knowledge translation may vary across different stakeholder groups. There are a large number of planned knowledge translation models, derived from different disciplinary, contextual (i.e., setting), and target audience viewpoints. Most of these suggest that planned knowledge translation for healthcare professionals and consumers is more likely to be successful if the choice of knowledge translation strategy is informed by an assessment of the

Cradle Cap (For Parents)

MedlinePlus

... Safe Videos for Educators Search English Español Cradle Cap (Infantile Seborrheic Dermatitis) KidsHealth / For Parents / Cradle Cap ( ... many babies develop called cradle cap. About Cradle Cap Cradle cap is the common term for seborrheic ...

Immunoprecipitation of Tri-methylated Capped RNA.

PubMed

Hayes, Karen E; Barr, Jamie A; Xie, Mingyi; Steitz, Joan A; Martinez, Ivan

2018-02-05

Cellular quiescence (also known as G 0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27 Kip1 . Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m 7 G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al ., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

PubMed Central

Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

2002-01-01

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

PubMed Central

Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

2012-01-01

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

Dopaminergic tone persistently regulates voltage-gated ion current densities through the D1R-PKA axis, RNA polymerase II transcription, RNAi, mTORC1, and translation

PubMed Central

Krenz, Wulf-Dieter C.; Parker, Anna R.; Rodgers, Edmund W.; Baro, Deborah J.

2014-01-01

Long-term intrinsic and synaptic plasticity must be coordinated to ensure stability and flexibility in neuronal circuits. Coordination might be achieved through shared transduction components. Dopamine (DA) is a well-established participant in many forms of long-term synaptic plasticity. Recent work indicates that DA is also involved in both activity-dependent and -independent forms of long-term intrinsic plasticity. We previously examined DA-enabled long-term intrinsic plasticity in a single identified neuron. The lateral pyloric (LP) neuron is a component of the pyloric network in the crustacean stomatogastric nervous system (STNS). LP expresses type 1 DA receptors (D1Rs). A 1 h bath application of 5 nM DA followed by washout produced a significant increase in the maximal conductance (Gmax) of the LP transient potassium current (IA) that peaked ~4 h after the start of DA application; furthermore, if a change in neuronal activity accompanied the DA application, then a persistent increase in the LP hyperpolarization activated current (Ih) was also observed. Here, we repeated these experiments with pharmacological and peptide inhibitors to determine the cellular processes and signaling proteins involved. We discovered that the persistent, DA-induced activity-independent (IA) and activity-dependent (Ih) changes in ionic conductances depended upon many of the same elements that enable long-term synaptic plasticity, including: the D1R-protein kinase A (PKA) axis, RNA polymerase II transcription, RNA interference (RNAi), and mechanistic target of rapamycin (mTOR)-dependent translation. We interpret the data to mean that increasing the tonic DA concentration enhances expression of a microRNA(s) (miRs), resulting in increased cap-dependent translation of an unidentified protein(s). PMID:24596543

Silicide formation process of Er films with Ta and TaN capping layers.

PubMed

Choi, Juyun; Choi, Seongheum; Kim, Jungwoo; Na, Sekwon; Lee, Hoo-Jeong; Lee, Seok-Hee; Kim, Hyoungsub

2013-12-11

The phase development and defect formation during the silicidation reaction of sputter-deposited Er films on Si with ∼20-nm-thick Ta and TaN capping layers were examined. TaN capping effectively prevented the oxygen incorporation from the annealing atmosphere, which resulted in complete conversion to the ErSi2-x phase. However, significant oxygen penetration through the Ta capping layer inhibited the ErSi2-x formation, and incurred the growth of several Er-Si-O phases, even consuming the ErSi2-x layer formed earlier. Both samples produced a number of small recessed defects at an early silicidation stage. However, large rectangular or square-shaped surface defects, which were either pitlike or pyramidal depending on the capping layer identity, were developed as the annealing temperature increased. The origin of different defect generation mechanisms was suggested based on the capping layer-dependent silicidation kinetics.

Template-dependent polypeptide synthesis in a factor- and energy-free translation system promoted by pyridine.

PubMed

Nitta, I; Ueda, T; Nojima, T; Watanabe, K

1995-10-01

We demonstrate here that a high concentration (40-70%) of pyridine, an aromatic tertiary amine catalyst, is able to promote translation on ribosomes without the presence of soluble protein factors or chemical energy sources. Compared with Monro's fragment reaction [Methods Enzymol. 20, 472-481 (1971)] which reflects only the peptidyltransferase step, this novel translation system can produce polypeptides with chain lengths of at least several tens of residues depending on the template RNA. In the presence of 60% pyridine, poly(U) and poly(UC) promoted incorporation of the respective amino acids, phenylalanine and serine-leucine, twofold, whereas poly(A) promoted the incorporation of lysine by only 25%. The degrees of polymerization of phenylalanine and lysine were up to the decamer and around 40mer, respectively. In poly(UC)-dependent oligo(serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products. This novel pyridine system evidently differs from the non-enzymatic translation system reported by Gavrilova and Spirin [FEBS Lett. 17, 324-326 (1971)]; the former system displays partial resistance toward deproteinization reagents such as SDS and proteinase K, whereas the latter system is completely sensitive.

5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

PubMed Central

Ogino, Minako

2017-01-01

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups

Rapid motor learning in the translational vestibulo-ocular reflex

NASA Technical Reports Server (NTRS)

Zhou, Wu; Weldon, Patrick; Tang, Bingfeng; King, W. M.; Shelhamer, M. J. (Principal Investigator)

2003-01-01

Motor learning was induced in the translational vestibulo-ocular reflex (TVOR) when monkeys were repeatedly subjected to a brief (0.5 sec) head translation while they tried to maintain binocular fixation on a visual target for juice rewards. If the target was world-fixed, the initial eye speed of the TVOR gradually increased; if the target was head-fixed, the initial eye speed of the TVOR gradually decreased. The rate of learning acquisition was very rapid, with a time constant of approximately 100 trials, which was equivalent to <1 min of accumulated stimulation. These learned changes were consolidated over >or=1 d without any reinforcement, indicating induction of long-term synaptic plasticity. Although the learning generalized to targets with different viewing distances and to head translations with different accelerations, it was highly specific for the particular combination of head motion and evoked eye movement associated with the training. For example, it was specific to the modality of the stimulus (translation vs rotation) and the direction of the evoked eye movement in the training. Furthermore, when one eye was aligned with the heading direction so that it remained motionless during training, learning was not expressed in this eye, but only in the other nonaligned eye. These specificities show that the learning sites are neither in the sensory nor the motor limb of the reflex but in the sensory-motor transformation stage of the reflex. The dependence of the learning on both head motion and evoked eye movement suggests that Hebbian learning may be one of the underlying cellular mechanisms.

No Escaping the Rat Race: Simulated Night Shift Work Alters the Time-of-Day Variation in BMAL1 Translational Activity in the Prefrontal Cortex.

PubMed

Marti, Andrea R; Patil, Sudarshan; Mrdalj, Jelena; Meerlo, Peter; Skrede, Silje; Pallesen, Ståle; Pedersen, Torhild T; Bramham, Clive R; Grønli, Janne

2017-01-01

, together with significant reduction in the synaptic plasticity associated protein activity-regulatedcytoskeleton-associated protein (Arc). Our results indicate considerable time-of-day and brain-region specific variation in cap-dependent translation initiation. We concludethat simulated night shift work in rats disrupts the pathways regulating the circadian component of the translation of mRNA in the PFC, and that this may partly explain impaired waking function during night shift work.

When a text is translated does the complexity of its vocabulary change? Translations and target readerships.

PubMed

Rêgo, Hênio Henrique Aragão; Braunstein, Lidia A; D'Agostino, Gregorio; Stanley, H Eugene; Miyazima, Sasuke

2014-01-01

In linguistic studies, the academic level of the vocabulary in a text can be described in terms of statistical physics by using a "temperature" concept related to the text's word-frequency distribution. We propose a "comparative thermo-linguistic" technique to analyze the vocabulary of a text to determine its academic level and its target readership in any given language. We apply this technique to a large number of books by several authors and examine how the vocabulary of a text changes when it is translated from one language to another. Unlike the uniform results produced using the Zipf law, using our "word energy" distribution technique we find variations in the power-law behavior. We also examine some common features that span across languages and identify some intriguing questions concerning how to determine when a text is suitable for its intended readership.

Monitoring Auroral Electrojet from Polar Cap Stations

NASA Astrophysics Data System (ADS)

Tan, A.; Lyatsky, W.; Lyatskaya, S.

2004-12-01

The auroral electrojet AL and AE geomagnetic activity indices are important for monitoring geomagnetic substorms. In the northern hemisphere these indices are derived from measurements at a set of geomagnetic observatories located in the auroral zone. In the southern hemisphere the major portion of the auroral zone is located on the ocean; this does not allow us to derive the auroral electrojet indices in the same way. We showed that monitoring the auroral electrojet is possible from magnetic field measurements at polar cap stations. For this purpose we used hourly values of geomagnetic field variations at four polar cap stations, distributed along polar cap boundary and occupying a longitudinal sector of about 14 hours, and calculated mean values of the total magnetic field disturbance T = (X2 + Y2 + Z2)1/2 where X, Y, and Z are geomagnetic field components measured at these polar cap stations. The set of the obtained values were called the T index. This index has a clear physical mining: it is the summary of geomagnetic disturbance in all three components averaged over the polar cap boundary. We found that correlation coefficients for the dependence of the T index on both AL and AE indices are as high as ~0.9 and higher. The high correlation of the T index with the AL and AE indices takes place for any UT hour when the stations were located at the night side. The T index further shows good correlation with solar wind parameters: the correlation coefficient for the dependence of the T index on the solar wind-geomagnetic activity coupling function is ~0.8 and higher, which is close to the correlation coefficient for AL index. The T index may be especially important in the cases when ground-based measurements in the auroral zone are impossible as in the southern hemisphere.

Rilonacept for the treatment of cryopyrin-associated periodic syndromes (CAPS).

PubMed

Hoffman, Hal M

2009-04-01

Cryopyrin-associated periodic syndromes (CAPS) encompass a group of rare inherited, autoinflammatory disorders that represent a spectrum of one disease with varying degrees of severity. Until recently, there was no effective treatment for CAPS, but identification of the genetic basis of CAPS highlighted the pathogenic role of IL-1beta. Rilonacept is a recently FDA approved biologic therapy for CAPS with high affinity for IL-1beta. Limited pharmacological data has been reported to date. A review of the phamacokinetics and pharmacodynamics data as well as the results of a pilot study and Phase III placebo-controlled trials of rilonacept in CAPS. Unpublished data on an open-label extension study in adult and pediatric subjects is also reviewed. Rilonacept produced rapid and profound improvements in symptoms and also reduced high-sesitivity C-reactive protein levels and normalized elevated serum amyloid A concentrations, an important risk factor for amyloidosis. The primary adverse events were injection- site reactions and upper respiratory tract infections. Rilonacept, the only IL-1 Trap, is the first of many novel IL-1-targeted therapies being developed. In a very short time it has changed the lives of CAPS patients.

Human ocular responses to translation of the observer and of the scene: dependence on viewing distance.

PubMed

Busettini, C; Miles, F A; Schwarz, U; Carl, J R

1994-01-01

Recent experiments on monkeys have indicated that the eye movements induced by brief translation of either the observer or the visual scene are a linear function of the inverse of the viewing distance. For the movements of the observer, the room was dark and responses were attributed to a translational vestibulo-ocular reflex (TVOR) that senses the motion through the otolith organs; for the movements of the scene, which elicit ocular following, the scene was projected and adjusted in size and speed so that the retinal stimulation was the same at all distances. The shared dependence on viewing distance was consistent with the hypothesis that the TVOR and ocular following are synergistic and share central pathways. The present experiments looked for such dependencies on viewing distance in human subjects. When briefly accelerated along the interaural axis in the dark, human subjects generated compensatory eye movements that were also a linear function of the inverse of the viewing distance to a previously fixated target. These responses, which were attributed to the TVOR, were somewhat weaker than those previously recorded from monkeys using similar methods. When human subjects faced a tangent screen onto which patterned images were projected, brief motion of those images evoked ocular following responses that showed statistically significant dependence on viewing distance only with low-speed stimuli (10 degrees/s). This dependence was at best weak and in the reverse direction of that seen with the TVOR, i.e., responses increased as viewing distance increased. We suggest that in generating an internal estimate of viewing distance subjects may have used a confounding cue in the ocular-following paradigm--the size of the projected scene--which was varied directly with the viewing distance in these experiments (in order to preserve the size of the retinal image). When movements of the subject were randomly interleaved with the movements of the scene--to encourage the

Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma

PubMed Central

Yang, Qingshan; Chen, Lisa S.; Neelapu, Sattva S.; Miranda, Roberto N.; Medeiros, L. Jeffrey

2012-01-01

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small mol-ecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL. PMID:22955922

Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma.

PubMed

Yang, Qingshan; Chen, Lisa S; Neelapu, Sattva S; Miranda, Roberto N; Medeiros, L Jeffrey; Gandhi, Varsha

2012-10-25

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small molecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL.

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

PubMed Central

Pelletier, J; Kaplan, G; Racaniello, V R; Sonenberg, N

1988-01-01

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction. Images PMID:2836606

Plant Translation Factors and Virus Resistance

PubMed Central

Sanfaçon, Hélène

2015-01-01

Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable. PMID:26114476

Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.

PubMed

Pelletier, Jerry; Graff, Jeremy; Ruggero, Davide; Sonenberg, Nahum

2015-01-15

Elevated protein synthesis is an important feature of many cancer cells and often arises as a consequence of increased signaling flux channeled to eukaryotic initiation factor 4F (eIF4F), the key regulator of the mRNA-ribosome recruitment phase of translation initiation. In many cellular and preclinical models of cancer, eIF4F deregulation results in changes in translational efficiency of specific mRNA classes. Importantly, many of these mRNAs code for proteins that potently regulate critical cellular processes, such as cell growth and proliferation, enhanced cell survival and cell migration that ultimately impinge on several hallmarks of cancer, including increased angiogenesis, deregulated growth control, enhanced cellular survival, epithelial-to-mesenchymal transition, invasion, and metastasis. By being positioned as the molecular nexus downstream of key oncogenic signaling pathways (e.g., Ras, PI3K/AKT/TOR, and MYC), eIF4F serves as a direct link between important steps in cancer development and translation initiation. Identification of mRNAs particularly responsive to elevated eIF4F activity that typifies tumorigenesis underscores the critical role of eIF4F in cancer and raises the exciting possibility of developing new-in-class small molecules targeting translation initiation as antineoplastic agents. ©2014 American Association for Cancer Research.

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

PubMed

Ochnik, Aleksandra M; Peterson, Mark S; Avdulov, Svetlana V; Oh, Annabell S; Bitterman, Peter B; Yee, Douglas

2016-02-01

Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Translating Alcohol Research

PubMed Central

Batman, Angela M.; Miles, Michael F.

2015-01-01

Alcohol use disorder (AUD) and its sequelae impose a major burden on the public health of the United States, and adequate long-term control of this disorder has not been achieved. Molecular and behavioral basic science research findings are providing the groundwork for understanding the mechanisms underlying AUD and have identified multiple candidate targets for ongoing clinical trials. However, the translation of basic research or clinical findings into improved therapeutic approaches for AUD must become more efficient. Translational research is a multistage process of streamlining the movement of basic biomedical research findings into clinical research and then to the clinical target populations. This process demands efficient bidirectional communication across basic, applied, and clinical science as well as with clinical practitioners. Ongoing work suggests rapid progress is being made with an evolving translational framework within the alcohol research field. This is helped by multiple interdisciplinary collaborative research structures that have been developed to advance translational work on AUD. Moreover, the integration of systems biology approaches with collaborative clinical studies may yield novel insights for future translational success. Finally, appreciation of genetic variation in pharmacological or behavioral treatment responses and optimal communication from bench to bedside and back may strengthen the success of translational research applications to AUD. PMID:26259085

CPI motif interaction is necessary for capping protein function in cells

PubMed Central

Edwards, Marc; McConnell, Patrick; Schafer, Dorothy A.; Cooper, John A.

2015-01-01

Capping protein (CP) has critical roles in actin assembly in vivo and in vitro. CP binds with high affinity to the barbed end of actin filaments, blocking the addition and loss of actin subunits. Heretofore, models for actin assembly in cells generally assumed that CP is constitutively active, diffusing freely to find and cap barbed ends. However, CP can be regulated by binding of the ‘capping protein interaction' (CPI) motif, found in a diverse and otherwise unrelated set of proteins that decreases, but does not abolish, the actin-capping activity of CP and promotes uncapping in biochemical experiments. Here, we report that CP localization and the ability of CP to function in cells requires interaction with a CPI-motif-containing protein. Our discovery shows that cells target and/or modulate the capping activity of CP via CPI motif interactions in order for CP to localize and function in cells. PMID:26412145

Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress.

PubMed

Sonobe, Yoshifumi; Ghadge, Ghanashyam; Masaki, Katsuhisa; Sendoel, Ataman; Fuchs, Elaine; Roos, Raymond P

2018-08-01

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G 4 C 2 construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G 4 C 2 constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle. Copyright © 2018 Elsevier Inc. All rights reserved.

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

PubMed Central

Korrapati, Anil Babu; Swaminathan, Gokul; Singh, Aarti; Khanna, Navin; Swaminathan, Sathyamangalam

2012-01-01

Background Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. PMID:22848770

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

PubMed

Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J

2017-01-01

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity.

PubMed

Ebbensgaard, Anna; Mordhorst, Hanne; Overgaard, Michael Toft; Aarestrup, Frank Møller; Hansen, Egon Bech

2018-01-01

Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.

Year-to-year instability of the Mars south polar cap

NASA Technical Reports Server (NTRS)

Jakosky, Bruce M.; Haberle, Robert M.

1990-01-01

Models of the Mars polar cap energy balance suggest that the south cap has two possible configurations at the current epoch. It can be covered all year by CO2 frost, as was observed by the Viking spacecraft, or the CO2 frost can disappear at midsummer to expose an underlying surface. The difference between the two states depends on the role of energy conducted into the subsurface. If the cap was exposed the previous year, conducted energy will cause less CO2 frost to condense in winter, and the frost will disappear completely in summer. If the cap was covered the previous year, there will be no conducted energy, and it will stay covered again. Subtle effects in the atmosphere can perturb the cap energy balance sufficiently to cause the cap to jump between states. This behavior might provide an explanation for the large southern summer water vapor abundances observed in 1969. It also explains the observed differences between the north and the south residual caps as resulting in part from a difference in the role in the annual energy balance of energy conducted into the subsurface.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

PubMed

Vvedenskaya, Irina O; Bird, Jeremy G; Zhang, Yuanchao; Zhang, Yu; Jiao, Xinfu; Barvík, Ivan; Krásný, Libor; Kiledjian, Megerditch; Taylor, Deanne M; Ebright, Richard H; Nickels, Bryce E

2018-05-03

Nucleoside-containing metabolites such as NAD + can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD + capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD + capping and define a consensus promoter sequence for NAD + capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD + -capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD + and provide a general method for analysis of NCIN capping in vitro and in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

When a Text Is Translated Does the Complexity of Its Vocabulary Change? Translations and Target Readerships

PubMed Central

Rêgo, Hênio Henrique Aragão; Braunstein, Lidia A.; D′Agostino, Gregorio; Stanley, H. Eugene; Miyazima, Sasuke

2014-01-01

In linguistic studies, the academic level of the vocabulary in a text can be described in terms of statistical physics by using a “temperature” concept related to the text's word-frequency distribution. We propose a “comparative thermo-linguistic” technique to analyze the vocabulary of a text to determine its academic level and its target readership in any given language. We apply this technique to a large number of books by several authors and examine how the vocabulary of a text changes when it is translated from one language to another. Unlike the uniform results produced using the Zipf law, using our “word energy” distribution technique we find variations in the power-law behavior. We also examine some common features that span across languages and identify some intriguing questions concerning how to determine when a text is suitable for its intended readership. PMID:25353343

Sex-Dependent Effects of Cannabis and Cannabinoids: A Translational Perspective.

PubMed

Cooper, Ziva D; Craft, Rebecca M

2018-01-01

Recent policy changes have led to significant increases in the use of cannabis for both medical and recreational purposes. Although men are more likely to endorse past month cannabis use and are more frequently diagnosed with Cannabis Use Disorder relative to women, a growing proportion of medical cannabis users are reported to be women. The increased popularity of cannabis for medical purposes and the narrowing gap in prevalence of use between men and women raises questions regarding sex-dependent effects related to therapeutic efficacy and negative health effects of cannabis and cannabinoids. The objective of this review is to provide a translational perspective on the sex-dependent effects of cannabis and cannabinoids by synthesizing findings from preclinical and clinical studies focused on sex comparisons of their therapeutic potential and abuse liability, two specific areas that are of significant public health relevance. Hormonal and pharmacological mechanisms that may underlie sex differences in the effects of cannabis and cannabinoids are highlighted.

Cradle Cap: Treatment

MedlinePlus

Cradle cap Treatment Cradle cap usually doesn't require medical treatment. It clears up on its own within a few months. In the meantime, wash ... tips can help you control and manage cradle cap. Gently rub your baby's scalp with your fingers ...

Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase, Germ Line Development Defective-2*

PubMed Central

Cragle, Chad; MacNicol, Angus M.

2014-01-01

The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation. PMID:24644291

Tracking the Martian CO2 Polar Ice Caps in Infrared Images

NASA Technical Reports Server (NTRS)

Wagstaff, Kiri L.; Castano, Rebecca; Chien, Steve

2006-01-01

Researchers at NASA s Jet Propulsion Laboratory have developed a method for automatically tracking the polar caps on Mars as they advance and recede each year (see figure). The seasonal Mars polar caps are composed mainly of CO2 ice and are therefore cold enough to stand out clearly in infrared data collected by the Thermal Emission Imaging System (THEMIS) onboard the Mars Odyssey spacecraft. The Bimodal Image Temperature (BIT) histogram analysis algorithm analyzes raw, uncalibrated data to identify images that contain both "cold" ("polar cap") and "warm" ("not polar cap") pixels. The algorithm dynamically identifies the temperature that separates these two regions. This flexibility is critical, because in the absence of any calibration, the threshold temperature can vary significantly from image to image. Using the identified threshold, the algorithm classifies each pixel in the image as "polar cap" or "not polar cap," then identifies the image row that contains the spatial transition from "polar cap" to "not polar cap." While this method is useful for analyzing data that has already been returned by THEMIS, it has even more significance with respect to data that has not yet been collected. Instead of seeking the polar cap only in specific, targeted images, the simplicity and efficiency of this method makes it feasible for direct, onboard use. That is, THEMIS could continuously monitor its observations for any detections of the polar-cap edge, producing detections over a wide range of spatial and temporal conditions. This effort can greatly contribute to our understanding of long-term climatic change on Mars.

Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets

PubMed Central

Stadler, Michael; Artiles, Karen; Pak, Julia; Fire, Andrew

2012-01-01

miRNAs are post-transcriptional regulators of gene activity that reduce protein accumulation from target mRNAs. Elucidating precise molecular effects that animal miRNAs have on target transcripts has proven complex, with varied evidence indicating that miRNA regulation may produce different molecular outcomes in different species, systems, and/or physiological conditions. Here we use high-throughput ribosome profiling to analyze detailed translational parameters for five well-studied targets of miRNAs that regulate C. elegans developmental timing. For two targets of the miRNA lin-4 (lin-14 and lin-28), functional down-regulation was associated with decreases in both overall mRNA abundance and ribosome loading; however, these changes were of substantially smaller magnitude than corresponding changes observed in protein abundance. For three functional targets of the let-7 miRNA family for which down-regulation is critical in temporal progression of the animal (daf-12, hbl-1, and lin-41), we observed only modest changes in mRNA abundance and ribosome loading. lin-41 provides a striking example in that populations of ribosome-protected fragments from this gene remained essentially unchanged during the L3–L4 time interval when lin-41 activity is substantially down-regulated by let-7. Spectra of ribosomal positions were also examined for the five lin-4 and let-7 target mRNAs as a function of developmental time, with no indication of miRNA-induced ribosomal drop-off or significant pauses in translation. These data are consistent with models in which physiological regulation by this set of C. elegans miRNAs derives from combinatorial effects including suppressed recruitment/activation of translational machinery, compromised stability of target messages, and post- or peri-translational effects on lifetimes of polypeptide products. PMID:22855835

Excessive Cap-dependent Translation as a Molecular Mechanism Underlying ASD

DTIC Science & Technology

2014-10-01

LSD acp HDB df SFO PVA S1FL I BSTMPM BSTMPI BSTMPL MCPO VP BSTLP...LGP Cl Pir VEn ACo SCh LOT cg vhc sm st ec ic rf I f IPACL AAV S1DZ TS SFi IPACM AC MPOL MPOM LSD acp HDB df SFO PVA S1FL I BSTMPM BSTMPI BSTMPL MCPO VP...Sweet, H.McDermid, S. R. Shaw , J. Claytor, J. Willis, D. P. Kelly, 22q13 deletion syndrome. Am. J. Med. Genet. 101, 91–99 (2001). 90. S. U. Dhar,

Paleo ice-cap surfaces and extents

NASA Astrophysics Data System (ADS)

Gillespie, A.; Pieri, D.

2008-12-01

The distribution, equilibrium-line altitude (ELA) and timing of Pleistocene alpine glaciers are used to constrain paleoclimatic reconstructions. Attention has largely focused on the geomorphic evidence for the former presence of simple valley glaciers; paleo alpine ice caps and their outlet glaciers have proven to be more problematical. This is especially so in the remote continental interior of Asia, where the research invested in the Alps or Rocky Mountains has yet to be duplicated. Even the putative existence and size of paleo ice caps in Tibet and the Kyrgyz Tien Shan is controversial. Remote sensing offers the opportunity to assess vast tracts of land quickly, with images and co-registered digital elevation models (DEMs) offering the most information for studies of paleoglaciers. We pose several questions: (1) With what confidence can nunataks be identified remotely? (2) What insights do their physiographic characteristics offer? (3) What characteristics of the bed of a paleo ice cap can be used to identify its former presence remotely? and (4) Can the geomorphic signatures of the edges of paleo ice caps be recognized and mapped? Reconstruction of the top surface of a paleo ice cap depends on the recognition of nunataks, typically rougher at 1 m to 100 m scales than their surroundings. Nunataks in southern Siberia are commonly notched by multiple sub- horizontal bedrock terraces. These step terraces appear to originate from freeze-thaw action on the rock-ice interface during periods of stability, and presence of multiple terraces suggests stepwise lowering of ice surfaces during deglaciation. An older generation of step-terraced nunataks, distinguished by degraded and eroded terraces, delineates a larger paleo ice cap in the Sayan Range (Siberian - Mongolian border) that significantly pre-dates the last glacial maximum (LGM). Large ice caps can experience pressure melting at their base and can manifest ice streams within the ice cap. Valleys left behind differ

Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Richter, S.; Zhang, R.

2009-09-04

Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamatemore » binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.« less

Establishment of chronic hepatitis C virus infection: Translational evasion of oxidative defence

PubMed Central

Chan, Shiu-Wan

2014-01-01

Hepatitis C virus (HCV) causes a clinically important disease affecting 3% of the world population. HCV is a single-stranded, positive-sense RNA virus belonging to the genus Hepacivirus within the Flaviviridae family. The virus establishes a chronic infection in the face of an active host oxidative defence, thus adaptation to oxidative stress is key to virus survival. Being a small RNA virus with a limited genomic capacity, we speculate that HCV deploys a different strategy to evade host oxidative defence. Instead of counteracting oxidative stress, it utilizes oxidative stress to facilitate its own survival. Translation is the first step in the replication of a plus strand RNA virus so it would make sense if the virus can exploit the host oxidative defence in facilitating this very first step. This is particularly true when HCV utilizes an internal ribosome entry site element in translation, which is distinctive from that of cap-dependent translation of the vast majority of cellular genes, thus allowing selective translation of genes under conditions when global protein synthesis is compromised. Indeed, we were the first to show that HCV translation was stimulated by an important pro-oxidant-hydrogen peroxide in hepatocytes, suggesting that HCV is able to adapt to and utilize the host anti-viral response to facilitate its own translation thus allowing the virus to thrive under oxidative stress condition to establish chronicity. Understanding how HCV translation is regulated under oxidative stress condition will advance our knowledge on how HCV establishes chronicity. As chronicity is the initiator step in disease progression this will eventually lead to a better understanding of pathogenicity, which is particularly relevant to the development of anti-virals and improved treatments of HCV patients using anti-oxidants. PMID:24659872

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

PubMed Central

Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

2004-01-01

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

The scanning model for translation: an update

PubMed Central

1989-01-01

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained. PMID:2645293

Alternative Ways to Think about Cellular Internal Ribosome Entry*

PubMed Central

Gilbert, Wendy V.

2010-01-01

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of “cap-dependent” and “IRES-dependent” should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation. PMID:20576611

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

PubMed

Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

2015-09-22

Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

Molecular basis for erythromycin-dependent ribosome-stalling during translation of the ErmBL leader peptide

PubMed Central

Arenz, Stefan; Ramu, Haripriya; Gupta, Pulkit; Berninghausen, Otto; Beckmann, Roland; Vázquez-Laslop, Nora; Mankin, Alexander S.; Wilson, Daniel N.

2014-01-01

In bacteria, ribosome-stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase center (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural bases for understanding how the drug and the nascent peptide cooperate to inhibit peptide-bond formation and induce translation arrest. PMID:24662426

UC/MALDI-MS analysis of HDL; evidence for density-dependent post-translational modifications

NASA Astrophysics Data System (ADS)

Johnson, Jeffery D.; Henriquez, Ronald R.; Tichy, Shane E.; Russell, David H.; McNeal, Catherine J.; Macfarlane, Ronald D.

2007-12-01

The purpose of this study is to determine whether the nature of the post-translational modifications of the major apolipoproteins of HDL is different for density-distinct subclasses. These subclasses were separated by ultracentrifugation using a novel density-forming solute to yield a high-resolution separation. The serum of two subjects, a control with a normolipidemic profile and a subject with diagnosed cardiovascular disease, was studied. Aliquots of three HDL subclasses were analyzed by MALDI and considerable differences were seen when comparing density-distinct subclasses and also when comparing the two subjects. A detailed analysis of the post-translational modification pattern of apoA-1 shows evidence of considerable protease activity, particularly in the more dense fractions. We conclude that part of the heterogeneity of the population of HDL particles is due to density-dependent protease activity.

Assessing the Role of Capping Molecules in Controlling Aggregative Growth of Gold Nanoparticles in Heated Solution.

PubMed

Cheng, Han-Wen; Schadt, Mark J; Zhong, Chuan-Jian

2016-01-01

This report describes findings of an investigation of the role of capping molecules in the size growth in the aggregative growth of pre-formed small-sized gold nanoparticles capped with alkanethiolate monolayers toward monodispersed larger sizes. The size controllability depends on the thiolate chain length and concentration in the thermal solution. The size evolution in solution at different concentrations of alkanethiols is analyzed in relation to adsorption isotherms and cohesive energy. The size dependence on thiolate chain length is also analyzed by considering the cohesive energy of the capping molecules, revealing the importance of cohesive energy in the capping structure. Theoretical and experimental comparisons of the surface plasmonic resonance optical properties have also provided new insights into the mechanism, thus enabling the exploitation of size-dependent nanoscale properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of cap lamp and laser illumination for detecting visual escape cues in smoke

PubMed Central

Lutz, T.J.; Sammarco, J.J.; Srednicki, J.R.; Gallagher, S.

2015-01-01

The Illuminating Engineering Society of North America reports that an underground mine is the most difficult environment to illuminate (Rea, 2000). Researchers at the U.S. National Institute for Occupational Safety and Health (NIOSH) Office of Mine Safety and Health Research (OMSHR) are conducting ongoing studies designed to explore different lighting technologies for improving mine safety. Underground miners use different visual cues to escape from a smoke-filled environment. Primary and secondary escapeways are marked with reflective ceiling tags of various colors. Miners also look for mine rail tracks. The main objective of this paper is to compare different lighting types and ceiling tag colors to differentiate what works best in a smoke-filled environment. Various cap lamps (LED and incandescent) and lasers (red, blue, green) were compared to see which options resulted in the longest detection distances for red, green and blue reflective markers and a section of mine rail track. All targets advanced toward the human subject inside of a smoke-filled room to simulate the subject walking in a mine environment. Detection distances were recorded and analyzed to find the best cap lamp, laser color and target color in a smoke environment. Results show that cap lamp, laser color and target color do make a difference in detection distances and are perceived differently based on subject age. Cap lamps were superior to lasers in all circumstances of ceiling tag detection, with the exception of the green laser. The incandescent cap lamp worked best in the simulated smoke compared to the LED cap lamps. The green laser was the best color for detecting the tags and track compared to the red and blue lasers. The green tags were the easiest color to detect on the ceiling. On average, the track was easier for the subjects to detect than the ceiling tags. PMID:26236146

Comparison of cap lamp and laser illumination for detecting visual escape cues in smoke.

PubMed

Lutz, T J; Sammarco, J J; Srednicki, J R; Gallagher, S

The Illuminating Engineering Society of North America reports that an underground mine is the most difficult environment to illuminate (Rea, 2000). Researchers at the U.S. National Institute for Occupational Safety and Health (NIOSH) Office of Mine Safety and Health Research (OMSHR) are conducting ongoing studies designed to explore different lighting technologies for improving mine safety. Underground miners use different visual cues to escape from a smoke-filled environment. Primary and secondary escapeways are marked with reflective ceiling tags of various colors. Miners also look for mine rail tracks. The main objective of this paper is to compare different lighting types and ceiling tag colors to differentiate what works best in a smoke-filled environment. Various cap lamps (LED and incandescent) and lasers (red, blue, green) were compared to see which options resulted in the longest detection distances for red, green and blue reflective markers and a section of mine rail track. All targets advanced toward the human subject inside of a smoke-filled room to simulate the subject walking in a mine environment. Detection distances were recorded and analyzed to find the best cap lamp, laser color and target color in a smoke environment. Results show that cap lamp, laser color and target color do make a difference in detection distances and are perceived differently based on subject age. Cap lamps were superior to lasers in all circumstances of ceiling tag detection, with the exception of the green laser. The incandescent cap lamp worked best in the simulated smoke compared to the LED cap lamps. The green laser was the best color for detecting the tags and track compared to the red and blue lasers. The green tags were the easiest color to detect on the ceiling. On average, the track was easier for the subjects to detect than the ceiling tags.

Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

PubMed

Lee, Hwa-Rim; Kim, Tae-Don; Kim, Hyo-Jin; Jung, Youngseob; Lee, Dohyun; Lee, Kyung-Ha; Kim, Do-Yeon; Woo, Kyung-Chul; Kim, Kyong-Tai

2015-11-01

Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

Cognate status and cross-script translation priming.

PubMed

Voga, Madeleine; Grainger, Jonathan

2007-07-01

Greek-French bilinguals were tested in three masked priming experiments with Greek primes and French targets. Related primes were the translation equivalents of target words, morphologically related to targets, or phonologically related to targets. In Experiment 1, cognate translation equivalents (phonologically similar translations) showed facilitatory priming, relative to matched phonologically related primes, in conditions in which morphologically related primes showed no effect (50-msec prime exposure). Cross-language morphological priming emerged at longer prime exposure durations (66 msec), but cognate primes continued to generate more priming than did those in the morphological condition. In Experiments 2 and 3, the level of phonological overlap across translation equivalents was varied, and priming effects were measured against those for matched phonologically related primes and those in an unrelated prime condition. When measured against the unrelated baseline, cognate primes showed the typical advantage over noncognate primes. However, this cognate advantage disappeared when priming was measured against the phonologically related prime condition. The results are discussed in terms of how translation equivalents are represented in bilingual memory.

Proteomic analysis of Medulloblastoma reveals functional biology with translational potential.

PubMed

Rivero-Hinojosa, Samuel; Lau, Ling San; Stampar, Mojca; Staal, Jerome; Zhang, Huizhen; Gordish-Dressman, Heather; Northcott, Paul A; Pfister, Stefan M; Taylor, Michael D; Brown, Kristy J; Rood, Brian R

2018-06-07

Genomic characterization has begun to redefine diagnostic classifications of cancers. However, it remains a challenge to infer disease phenotypes from genomic alterations alone. To help realize the promise of genomics, we have performed a quantitative proteomics investigation using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and 41 tissue samples spanning the 4 genomically based subgroups of medulloblastoma and control cerebellum. We have identified and quantitated thousands of proteins across these groups and find that we are able to recapitulate the genomic subgroups based upon subgroup restricted and differentially abundant proteins while also identifying subgroup specific protein isoforms. Integrating our proteomic measurements with genomic data, we calculate a poor correlation between mRNA and protein abundance. Using EPIC 850 k methylation array data on the same tissues, we also investigate the influence of copy number alterations and DNA methylation on the proteome in an attempt to characterize the impact of these genetic features on the proteome. Reciprocally, we are able to use the proteome to identify which genomic alterations result in altered protein abundance and thus are most likely to impact biology. Finally, we are able to assemble protein-based pathways yielding potential avenues for clinical intervention. From these, we validate the EIF4F cap-dependent translation pathway as a novel druggable pathway in medulloblastoma. Thus, quantitative proteomics complements genomic platforms to yield a more complete understanding of functional tumor biology and identify novel therapeutic targets for medulloblastoma.

Carotid Plaque Morphological Classification Compared With Biomechanical Cap Stress: Implications for a Magnetic Resonance Imaging-Based Assessment.

PubMed

Gijsen, Frank J H; Nieuwstadt, Harm A; Wentzel, Jolanda J; Verhagen, Hence J M; van der Lugt, Aad; van der Steen, Antonius F W

2015-08-01

Two approaches to target plaque vulnerability-a histopathologic classification scheme and a biomechanical analysis-were compared and the implications for noninvasive risk stratification of carotid plaques using magnetic resonance imaging were assessed. Seventy-five histological plaque cross sections were obtained from carotid endarterectomy specimens from 34 patients (>70% stenosis) and subjected to both a Virmani histopathologic classification (thin fibrous cap atheroma with <0.2-mm cap thickness, presumed vulnerable) and a peak cap stress computation (<140 kPa: presumed stable; >300 kPa: presumed vulnerable). To demonstrate the implications for noninvasive plaque assessment, numeric simulations of a typical carotid magnetic resonance imaging protocol were performed (0.62×0.62 mm(2) in-plane acquired voxel size) and used to obtain the magnetic resonance imaging-based peak cap stress. Peak cap stress was generally associated with histological classification. However, only 16 of 25 plaque cross sections could be labeled as high-risk (peak cap stress>300 kPa and classified as a thin fibrous cap atheroma). Twenty-eight of 50 plaque cross sections could be labeled as low-risk (a peak cap stress<140 kPa and not a thin fibrous cap atheroma), leading to a κ=0.39. 31 plaques (41%) had a disagreement between both classifications. Because of the limited magnetic resonance imaging voxel size with regard to cap thickness, a noninvasive identification of only a group of low-risk, thick-cap plaques was reliable. Instead of trying to target only vulnerable plaques, a more reliable noninvasive identification of a select group of stable plaques with a thick cap and low stress might be a more fruitful approach to start reducing surgical interventions on carotid plaques. © 2015 American Heart Association, Inc.

Spatial frequency dependence of target signature for infrared performance modeling

NASA Astrophysics Data System (ADS)

Du Bosq, Todd; Olson, Jeffrey

2011-05-01

The standard model used to describe the performance of infrared imagers is the U.S. Army imaging system target acquisition model, based on the targeting task performance metric. The model is characterized by the resolution and sensitivity of the sensor as well as the contrast and task difficulty of the target set. The contrast of the target is defined as a spatial average contrast. The model treats the contrast of the target set as spatially white, or constant, over the bandlimit of the sensor. Previous experiments have shown that this assumption is valid under normal conditions and typical target sets. However, outside of these conditions, the treatment of target signature can become the limiting factor affecting model performance accuracy. This paper examines target signature more carefully. The spatial frequency dependence of the standard U.S. Army RDECOM CERDEC Night Vision 12 and 8 tracked vehicle target sets is described. The results of human perception experiments are modeled and evaluated using both frequency dependent and independent target signature definitions. Finally the function of task difficulty and its relationship to a target set is discussed.

One-dimensional cold cap model for melters with bubblers

DOE PAGES

Pokorny, Richard; Hilliard, Zachary J.; Dixon, Derek R.; ...

2015-07-28

The rate of glass production during vitrification in an all-electrical melter greatly impacts the cost and schedule of nuclear waste treatment and immobilization. The feed is charged to the melter on the top of the molten glass, where it forms a layer of reacting and melting material, called the cold cap. During the final stages of the batch-to-glass conversion process, gases evolved from reactions produce primary foam, the growth and collapse of which controls the glass production rate. The mathematical model of the cold cap was revised to include functional representation of primary foam behavior and to account for themore » dry cold cap surface. The melting rate is computed as a response to the dependence of the primary foam collapse temperature on the heating rate and melter operating conditions, including the effect of bubbling on the cold cap bottom and top surface temperatures. The simulation results are in good agreement with experimental data from laboratory-scale and pilot-scale melter studies. Lastly, the cold cap model will become part of the full three-dimensional mathematical model of the waste glass melter.« less

DEAD ZONE IN THE POLAR-CAP ACCELERATOR OF PULSARS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Alexander Y.; Beloborodov, Andrei M.

We study plasma flows above pulsar polar caps using time-dependent simulations of plasma particles in the self-consistent electric field. The flow behavior is controlled by the dimensionless parameter {alpha} = j/c{rho}{sub GJ}, where j is the electric current density and {rho}{sub GJ} is the Goldreich-Julian charge density. The region of the polar cap where 0 < {alpha} < 1 is a {sup d}ead zone{sup -}in this zone, particle acceleration is inefficient and pair creation is not expected even for young, rapidly rotating pulsars. Pulsars with polar caps near the rotation axis are predicted to have a hollow-cone structure of radiomore » emission, as the dead zone occupies the central part of the polar cap. Our results apply to charge-separated flows of electrons (j < 0) or ions (j > 0). In the latter case, we consider the possibility of a mixed flow consisting of different ion species, and observe the development of two-stream instability. The dead zone at the polar cap is essential for the development of an outer gap near the null surface {rho}{sub GJ} = 0.« less

Translation between representation languages

NASA Technical Reports Server (NTRS)

Vanbaalen, Jeffrey

1994-01-01

A capability for translating between representation languages is critical for effective knowledge base reuse. A translation technology for knowledge representation languages based on the use of an interlingua for communicating knowledge is described. The interlingua-based translation process consists of three major steps: translation from the source language into a subset of the interlingua, translation between subsets of the interlingua, and translation from a subset of the interlingua into the target language. The first translation step into the interlingua can typically be specified in the form of a grammar that describes how each top-level form in the source language translates into the interlingua. In cases where the source language does not have a declarative semantics, such a grammar is also a specification of a declarative semantics for the language. A methodology for building translators that is currently under development is described. A 'translator shell' based on this methodology is also under development. The shell has been used to build translators for multiple representation languages and those translators have successfully translated nontrivial knowledge bases.

Primate translational vestibuloocular reflexes. II. Version and vergence responses to fore-aft motion

NASA Technical Reports Server (NTRS)

McHenry, M. Q.; Angelaki, D. E.

2000-01-01

To maintain binocular fixation on near targets during fore-aft translational disturbances, largely disjunctive eye movements are elicited the amplitude and direction of which should be tuned to the horizontal and vertical eccentricities of the target. The eye movements generated during this task have been investigated here as trained rhesus monkeys fixated isovergence targets at different horizontal and vertical eccentricities during 10 Hz fore-aft oscillations. The elicited eye movements complied with the geometric requirements for binocular fixation, although not ideally. First, the corresponding vergence angle for which the movement of each eye would be compensatory was consistently less than that dictated by the actual fixation parameters. Second, the eye position with zero sensitivity to translation was not straight ahead, as geometrically required, but rather exhibited a systematic dependence on viewing distance and vergence angle. Third, responses were asymmetric, with gains being larger for abducting and downward compared with adducting and upward gaze directions, respectively. As frequency was varied between 4 and 12 Hz, responses exhibited high-pass filter properties with significant differences between abduction and adduction responses. As a result of these differences, vergence sensitivity increased as a function of frequency with a steeper slope than that of version. Despite largely undercompensatory version responses, vergence sensitivity was closer to ideal. Moreover, the observed dependence of vergence sensitivity on vergence angle, which was varied between 2.5 and 10 MA, was largely linear rather than quadratic (as geometrically predicted). We conclude that the spatial tuning of eye velocity sensitivity as a function of gaze and viewing distance follows the general geometric dependencies required for the maintenance of foveal visual acuity. However, systematic deviations from ideal behavior exist that might reflect asymmetric processing of

Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP

PubMed Central

Meier, Jan; Hovestadt, Volker; Zapatka, Marc; Pscherer, Armin; Lichter, Peter; Seiffert, Martina

2013-01-01

MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity. PMID:23673373

Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cauwe, Benedicte; Martens, Erik; Van den Steen, Philippe E.

2008-09-10

Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1more » was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.« less

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

PubMed

Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Regulation of D-cyclin translation inhibition in myeloma cells treated with mTOR inhibitors: Rationale for combined treatment with ERK inhibitors and rapamycin

PubMed Central

Frost, Patrick; Shi, Yijiang; Hoang, Bao; Gera, Joseph; Lichtenstein, Alan

2009-01-01

We have shown that heightened AKT activity sensitized multiple myeloma (MM) cells to the anti-tumor effects of the mTOR-inhibitor, CCI-779. To test the mechanism of AKT’s regulatory role, we stably transfected U266 MM cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analog, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mTOR inhibitors to downregulate D-cyclin expression was significantly greater in AKT-transfected MM cells, due in part, to AKT’s ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was demonstrated in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild type PTEN. As ERK/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells demonstrated significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. However, ectopic over-expression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high AKT” myeloma cells indicating that MEK/ERK activity was required but not sufficient for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in MM cells and ERK facilitates activity. PMID:19139116

The antioxidative and hepatoprotective effects comparison of Chinese angelica polysaccharide(CAP)and selenizing CAP (sCAP) in CCl4 induced hepatic injury mice.

PubMed

Gao, Zhenzhen; Zhang, Chao; Tian, Weijun; Liu, Kuanhui; Hou, Ranran; Yue, Chanjuan; Wu, Yi; Wang, Deyun; Liu, Jiaguo; Hu, Yuanliang; Yang, Ying

2017-04-01

Chinese angelica polysaccharides (CAP) and selenizing CAP (sCAP) were prepared and identified through FTIR and SEM observation. Their antioxidant activities in vitro and hepatoprotective effects in vivo were compared by free radical-scavenging tests or with CCl 4 -induced hepatic injury model mice. The results showed that for DPPH radical, superoxide anion and hydroxyl radical, the scavenging capabilities of sCAP were significantly stronger than those of CAP . In hepatic injury model mice, sCAP could significantly reduce ALT, AST and ALP contents and raised TP content in serum, significantly reduce MDA and ROS contents and raised SOD and T-AOC activities in liver homogenate in comparison with CAP; obviously relieve the pathological changes of liver and significantly inhibit the expressions of p-ERK, p-JNK and p-p38 protein as compared with those in model control group. These results indicate that selenylation modification can enhance the antioxidant and hepatoprotective actions of Chinese angelica polysaccharide. A action mechanism of sCAP is suppressing the protein expression of MAPK signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Translational PK-PD modelling of molecular target modulation for the AMPA receptor positive allosteric modulator Org 26576.

PubMed

Bursi, Roberta; Erdemli, Gul; Campbell, Robert; Hutmacher, Matthew M; Kerbusch, Thomas; Spanswick, David; Jeggo, Ross; Nations, Kari R; Dogterom, Peter; Schipper, Jacques; Shahid, Mohammed

2011-12-01

The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor potentiator Org 26576 represents an interesting pharmacological tool to evaluate the utility of glutamatergic enhancement towards the treatment of psychiatric disorders. In this study, a rat-human translational pharmacokinetic-pharmacodynamic (PK-PD) model of AMPA receptor modulation was used to predict human target engagement and inform dose selection in efficacy clinical trials. Modelling and simulation was applied to rat plasma and cerebrospinal fluid (CSF) pharmacokinetic and pharmacodynamic measurements to identify a target concentration (EC(80)) for AMPA receptor modulation. Human plasma pharmacokinetics was determined from 33 healthy volunteers and eight major depressive disorder patients. From four out of these eight patients, CSF PK was also determined. Simulations of human CSF levels were performed for several doses of Org 26576. Org 26576 (0.1-10 mg/kg, i.v.) potentiated rat hippocampal AMPA receptor responses in an exposure-dependant manner. The rat plasma and CSF PK data were fitted by one-compartment model each. The rat CSF PK-PD model yielded an EC(80) value of 593 ng/ml (90% confidence interval 406.8, 1,264.1). The human plasma and CSF PK data were simultaneously well described by a two-compartment model. Simulations showed that in humans at 100 mg QD, CSF levels of Org 26576 would exceed the EC(80) target concentration for about 2 h and that 400 mg BID would engage AMPA receptors for 24 h. The modelling approach provided useful insight on the likely human dose-molecular target engagement relationship for Org 26576. Based on the current analysis, 100 and 400 mg BID would be suitable to provide 'phasic' and 'continuous' AMPA receptor engagement, respectively.

Broad spectrum antiviral agent ribavirin inhibits capping of mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goswami, B.B.; Borek, E.; Sharma, O.K.

1979-08-13

Ribavirin (1-..beta..-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common target for a chemotherapeutic agent in both DNA and RNA viruses is the capping reaction of mRNAs which inter alia involves the formation of a guanine pyrophosphate structure at the 5' terminus by mRNA guanylyl transferase. We have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency ofmore » the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped.« less

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking.

PubMed

Miras, Manuel; Sempere, Raquel N; Kraft, Jelena J; Miller, W Allen; Aranda, Miguel A; Truniger, Veronica

2014-04-01

Many plant viruses depend on functional RNA elements, called 3'-UTR cap-independent translation enhancers (3'-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3'-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3'-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3'-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3'-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise nonsusceptible host. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking

PubMed Central

Miras, Manuel; Sempere, Raquel N.; Kraft, Jelena J.; Miller, W. Allen; Aranda, Miguel A.; Truniger, Veronica

2015-01-01

Summary Many plant viruses depend on functional RNA elements, called 3′-UTR cap-independent translation enhancers (3′-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3′-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3′-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3′-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3′-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise non-susceptible host. PMID:24372390

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

New inverse synthetic aperture radar algorithm for translational motion compensation

NASA Astrophysics Data System (ADS)

Bocker, Richard P.; Henderson, Thomas B.; Jones, Scott A.; Frieden, B. R.

1991-10-01

Inverse synthetic aperture radar (ISAR) is an imaging technique that shows real promise in classifying airborne targets in real time under all weather conditions. Over the past few years a large body of ISAR data has been collected and considerable effort has been expended to develop algorithms to form high-resolution images from this data. One important goal of workers in this field is to develop software that will do the best job of imaging under the widest range of conditions. The success of classifying targets using ISAR is predicated upon forming highly focused radar images of these targets. Efforts to develop highly focused imaging computer software have been challenging, mainly because the imaging depends on and is affected by the motion of the target, which in general is not precisely known. Specifically, the target generally has both rotational motion about some axis and translational motion as a whole with respect to the radar. The slant-range translational motion kinematic quantities must be first accurately estimated from the data and compensated before the image can be focused. Following slant-range motion compensation, the image is further focused by determining and correcting for target rotation. The use of the burst derivative measure is proposed as a means to improve the computational efficiency of currently used ISAR algorithms. The use of this measure in motion compensation ISAR algorithms for estimating the slant-range translational motion kinematic quantities of an uncooperative target is described. Preliminary tests have been performed on simulated as well as actual ISAR data using both a Sun 4 workstation and a parallel processing transputer array. Results indicate that the burst derivative measure gives significant improvement in processing speed over the traditional entropy measure now employed.

Dendritic Glutamate Receptor mRNAs Show Contingent Local Hotspot-Dependent Translational Dynamics

PubMed Central

Kim, Tae Kyung; Sul, Jai-Yoon; Helmfors, Henrik; Langel, Ulo; Kim, Junhyong; Eberwine, James

2014-01-01

SUMMARY Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s-3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite. PMID:24075992

Ellipsoidal corrections for geoid undulation computations using gravity anomalies in a cap

NASA Technical Reports Server (NTRS)

Rapp, R. H.

1981-01-01

Ellipsoidal correction terms have been derived for geoid undulation computations when the Stokes equation using gravity anomalies in a cap is combined with potential coefficient information. The correction terms are long wavelength and depend on the cap size in which its gravity anomalies are given. Using the regular Stokes equation, the maximum correction for a cap size of 20 deg is -33 cm, which reduces to -27 cm when the Stokes function is modified by subtracting the value of the Stokes function at the cap radius. Ellipsoidal correction terms were also derived for the well-known Marsh/Chang geoids. When no gravity was used, the correction could reach 101 cm, while for a cap size of 20 deg the maximum correction was -45 cm. Global correction maps are given for a number of different cases. For work requiring accurate geoid computations these correction terms should be applied.

A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted Molecular Imaging of Tumors in Living Subjects

PubMed Central

Gao, Jinhao; Chen, Kai; Luong, Richard; Bouley, Donna M.; Mao, Hua; Qiao, Tiecheng; Gambhir, Sanjiv S.; Cheng, Zhen

2011-01-01

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QDs as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding non-tumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD2 to integrin αvβ3–positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron based nanoprobes in the clinical setting in the near future. PMID:22172022

A novel clinically translatable fluorescent nanoparticle for targeted molecular imaging of tumors in living subjects.

PubMed

Gao, Jinhao; Chen, Kai; Luong, Richard; Bouley, Donna M; Mao, Hua; Qiao, Tiecheng; Gambhir, Sanjiv S; Cheng, Zhen

2012-01-11

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)β(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future. © 2011 American Chemical Society

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

PubMed Central

Eaton, Heather E.; Kobayashi, Takeshi; Dermody, Terence S.; Johnston, Randal N.

2017-01-01

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since

Reader, Text, Translation, and Interpretive Potentials.

ERIC Educational Resources Information Center

Dollerup, Cay; And Others

No matter what pains translators take to produce a target-language text "identical" to the source-language text, criticism and/or translation of an original literary work cannot be the same in different language communities. That translation may change potentialities in the textual experience is particularly obvious in literature with a…

Translation invariant time-dependent solutions to massive gravity II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mourad, J.; Steer, D.A., E-mail: mourad@apc.univ-paris7.fr, E-mail: steer@apc.univ-paris7.fr

2014-06-01

This paper is a sequel to JCAP 12 (2013) 004 and is also devoted to translation-invariant solutions of ghost-free massive gravity in its moving frame formulation. Here we consider a mass term which is linear in the vielbein (corresponding to a β{sub 3} term in the 4D metric formulation) in addition to the cosmological constant. We determine explicitly the constraints, and from the initial value formulation show that the time-dependent solutions can have singularities at a finite time. Although the constraints give, as in the β{sub 1} case, the correct number of degrees of freedom for a massive spin twomore » field, we show that the lapse function can change sign at a finite time causing a singular time evolution. This is very different to the β{sub 1} case where time evolution is always well defined. We conclude that the β{sub 3} mass term can be pathological and should be treated with care.« less

Incorporating 3D-printing technology in the design of head-caps and electrode drives for recording neurons in multiple brain regions

PubMed Central

DeLucca, Michael V.; Haufler, Darrell; Paré, Denis

2015-01-01

Recent advances in recording and computing hardware have enabled laboratories to record the electrical activity of multiple brain regions simultaneously. Lagging behind these technical advances, however, are the methods needed to rapidly produce microdrives and head-caps that can flexibly accommodate different recording configurations. Indeed, most available designs target single or adjacent brain regions, and, if multiple sites are targeted, specially constructed head-caps are used. Here, we present a novel design style, for both microdrives and head-caps, which takes advantage of three-dimensional printing technology. This design facilitates targeting of multiple brain regions in various configurations. Moreover, the parts are easily fabricated in large quantities, with only minor hand-tooling and finishing required. PMID:25652930

A network model of genomic hormone interactions underlying dementia and its translational validation through serendipitous off-target effect

PubMed Central

2013-01-01

Background While the majority of studies have focused on the association between sex hormones and dementia, emerging evidence supports the role of other hormone signals in increasing dementia risk. However, due to the lack of an integrated view on mechanistic interactions of hormone signaling pathways associated with dementia, molecular mechanisms through which hormones contribute to the increased risk of dementia has remained unclear and capacity of translating hormone signals to potential therapeutic and diagnostic applications in relation to dementia has been undervalued. Methods Using an integrative knowledge- and data-driven approach, a global hormone interaction network in the context of dementia was constructed, which was further filtered down to a model of convergent hormone signaling pathways. This model was evaluated for its biological and clinical relevance through pathway recovery test, evidence-based analysis, and biomarker-guided analysis. Translational validation of the model was performed using the proposed novel mechanism discovery approach based on ‘serendipitous off-target effects’. Results Our results reveal the existence of a well-connected hormone interaction network underlying dementia. Seven hormone signaling pathways converge at the core of the hormone interaction network, which are shown to be mechanistically linked to the risk of dementia. Amongst these pathways, estrogen signaling pathway takes the major part in the model and insulin signaling pathway is analyzed for its association to learning and memory functions. Validation of the model through serendipitous off-target effects suggests that hormone signaling pathways substantially contribute to the pathogenesis of dementia. Conclusions The integrated network model of hormone interactions underlying dementia may serve as an initial translational platform for identifying potential therapeutic targets and candidate biomarkers for dementia-spectrum disorders such as Alzheimer�

Direct colorimetric detection of unamplified pathogen DNA by dextrin-capped gold nanoparticles.

PubMed

Baetsen-Young, Amy M; Vasher, Matthew; Matta, Leann L; Colgan, Phil; Alocilja, Evangelyn C; Day, Brad

2018-03-15

The interaction between gold nanoparticles (AuNPs) and nucleic acids has facilitated a variety of diagnostic applications, with further diversification of synthesis match bio-applications while reducing biotoxicity. However, DNA interactions with unique surface capping agents have not been fully defined. Using dextrin-capped AuNPs (d-AuNPs), we have developed a novel unamplified genomic DNA (gDNA) nanosensor, exploiting dispersion and aggregation characteristics of d-AuNPs, in the presence of gDNA, for sequence-specific detection. We demonstrate that d-AuNPs are stable in a five-fold greater salt concentration than citrate-capped AuNPs and the d-AuNPs were stabilized by single stranded DNA probe (ssDNAp). However, in the elevated salt concentrations of the DNA detection assay, the target reactions were surprisingly further stabilized by the formation of a ssDNAp-target gDNA complex. The results presented herein lead us to propose a mechanism whereby genomic ssDNA secondary structure formation during ssDNAp-to-target gDNA binding enables d-AuNP stabilization in elevated ionic environments. Using the assay described herein, we were successful in detecting as little as 2.94 fM of pathogen DNA, and using crude extractions of a pathogen matrix, as few as 18 spores/µL. Copyright © 2017 Elsevier B.V. All rights reserved.

[CAP quality management system in clinical laboratory and its issue].

PubMed

Tazawa, Hiromitsu

2004-03-01

The CAP (College of American Pathologists) was established in 1962 and, at present, CAP-accredited laboratories include about 6000 institutions all over the world, mainly in the U.S. The essential purpose of CAP accreditation is high quality reservation and improvement of clinical laboratory services for patient care, and is based on seven points, listed below. (1) Establishment of a laboratory management program and laboratory techniques to assure accuracy and improve overall quality of laboratory services. (2) Maintenance and improvement of accuracy objectively by centering on a CAP survey. (3) Thoroughness in safety and health administration. (4) Reservation of the performance of laboratory services by personnel and proficiency management. (5) Provision of appropriate information to physicians, and contribution to improved quality of patient care by close communication with physicians (improvement in patient care). (6) Reduction of running costs and personnel costs based on evidence by employing the above-mentioned criteria. (7) Reduction of laboratory error. In the future, accreditation and/or certification by organizations such as CAP, ISO, etc., may become a requirement for providing any clinical laboratory services in Japan. Taking the essence of the CAP and the characteristics of the new international standard, ISO151589, into consideration, it is important to choose the best suited accreditation and/or certification depending of the purpose of clinical laboratory.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

PubMed Central

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angèle; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. PMID:21343906

The pharmaceutical vial capping process: Container closure systems, capping equipment, regulatory framework, and seal quality tests.

PubMed

Mathaes, Roman; Mahler, Hanns-Christian; Buettiker, Jean-Pierre; Roehl, Holger; Lam, Philippe; Brown, Helen; Luemkemann, Joerg; Adler, Michael; Huwyler, Joerg; Streubel, Alexander; Mohl, Silke

2016-02-01

Parenteral drug products are protected by appropriate primary packaging to protect against environmental factors, including potential microbial contamination during shelf life duration. The most commonly used CCS configuration for parenteral drug products is the glass vial, sealed with a rubber stopper and an aluminum crimp cap. In combination with an adequately designed and controlled aseptic fill/finish processes, a well-designed and characterized capping process is indispensable to ensure product quality and integrity and to minimize rejections during the manufacturing process. In this review, the health authority requirements and expectations related to container closure system quality and container closure integrity are summarized. The pharmaceutical vial, the rubber stopper, and the crimp cap are described. Different capping techniques are critically compared: The most common capping equipment with a rotating capping plate produces the lowest amount of particle. The strength and challenges of methods to control the capping process are discussed. The residual seal force method can characterize the capping process independent of the used capping equipment or CCS. We analyze the root causes of several cosmetic defects associated with the vial capping process. Copyright © 2015 Elsevier B.V. All rights reserved.

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

PubMed

Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

2016-07-01

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Lost in translation: assessing effectiveness of focus group questioning techniques to develop improved translation of terminology used in HIV prevention clinical trials.

PubMed

Mack, Natasha; Ramirez, Catalina B; Friedland, Barbara; Nnko, Soori

2013-01-01

Achieving participant comprehension has proven to be one of the most difficult, practical, and ethical challenges of HIV prevention clinical trials. It becomes even more challenging when local languages do not have equivalent scientific and technical vocabularies, rendering communication of scientific concepts in translated documents extremely difficult. Even when bilingual lexicons are developed, there is no guarantee that participants understand the terminology as translated. We conducted twelve focus groups with women of reproductive age in Mwanza, Tanzania to explore the effectiveness of four questioning techniques for: (1) assessing participants' familiarity with existing technical terms and concepts, (2) generating a list of acceptable technical and non-technical terms, (3) testing our definitions of technical terms, and (4) verifying participants' preferences for terms. Focus groups were transcribed, translated, and qualitatively analyzed. A translation process that uses all four questioning techniques in a step-wise approach is an effective way to establish a baseline understanding of participants' familiarity with research terms, to develop and test translatable definitions, and to identify participants' preferred terminology for international HIV clinical research. This may help to ensure that important concepts are not "lost in translation." The results emphasize the importance of using a variety of techniques depending on the level of participant familiarity with research concepts, the existence of colloquial or technical terms in the target language, and the inherent complexity of the terms.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

PubMed Central

Leen, Eoin N.; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C.; Matthews, Stephen J.; Goodfellow, Ian G.; Curry, Stephen

2016-01-01

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. PMID:26734730

Dysregulation of mTOR signaling in fragile X syndrome.

PubMed

Sharma, Ali; Hoeffer, Charles A; Takayasu, Yukihiro; Miyawaki, Takahiro; McBride, Sean M; Klann, Eric; Zukin, R Suzanne

2010-01-13

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.

PubMed

Garg, Seema; De, Arnab; Mozumdar, Subho

2015-05-01

Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future. © 2014 Wiley Periodicals, Inc.

CRISPRi-sRNA: Transcriptional-Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis.

PubMed

Cao, Yingxiu; Li, Xiaofei; Li, Feng; Song, Hao

2017-09-15

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

Prospects for detection of target-dependent annual modulation in direct dark matter searches

DOE PAGES

Nobile, Eugenio Del; Gelmini, Graciela B.; Witte, Samuel J.

2016-02-03

Earth's rotation about the Sun produces an annual modulation in the expected scattering rate at direct dark matter detection experiments. The annual modulation as a function of the recoil energy E R imparted by the dark matter particle to a target nucleus is expected to vary depending on the detector material. However, for most interactions a change of variables from E R to v min, the minimum speed a dark matter particle must have to impart a fixed E R to a target nucleus, produces an annual modulation independent of the target element. We recently showed that if the darkmore » matter-nucleus cross section contains a non-factorizable target and dark matter velocity dependence, the annual modulation as a function of v min can be target dependent. Here we examine more extensively the necessary conditions for target-dependent modulation, its observability in present-day experiments, and the extent to which putative signals could identify a dark matter-nucleus differential cross section with a non-factorizable dependence on the dark matter velocity.« less

Text Coherence in Translation

ERIC Educational Resources Information Center

Zheng, Yanping

2009-01-01

In the thesis a coherent text is defined as a continuity of senses of the outcome of combining concepts and relations into a network composed of knowledge space centered around main topics. And the author maintains that in order to obtain the coherence of a target language text from a source text during the process of translation, a translator can…

Incorporating 3D-printing technology in the design of head-caps and electrode drives for recording neurons in multiple brain regions.

PubMed

Headley, Drew B; DeLucca, Michael V; Haufler, Darrell; Paré, Denis

2015-04-01

Recent advances in recording and computing hardware have enabled laboratories to record the electrical activity of multiple brain regions simultaneously. Lagging behind these technical advances, however, are the methods needed to rapidly produce microdrives and head-caps that can flexibly accommodate different recording configurations. Indeed, most available designs target single or adjacent brain regions, and, if multiple sites are targeted, specially constructed head-caps are used. Here, we present a novel design style, for both microdrives and head-caps, which takes advantage of three-dimensional printing technology. This design facilitates targeting of multiple brain regions in various configurations. Moreover, the parts are easily fabricated in large quantities, with only minor hand-tooling and finishing required. Copyright © 2015 the American Physiological Society.

Impaired cytokine responses in patients with cryopyrin-associated periodic syndrome (CAPS)

PubMed Central

Haverkamp, M H; van de Vosse, E; Goldbach-Mansky, R; Holland, S M

2014-01-01

Cryopyrin-associated periodic syndrome (CAPS) is characterized by dysregulated inflammation with excessive interleukin (IL)-1β activation and secretion. Neonatal-onset multi-system inflammatory disease (NOMID) is the most severe form. We explored cytokine responses in 32 CAPS patients before and after IL-1β blocking therapy. We measured cytokines produced by activated peripheral blood monuclear cells (PBMCs) from treated and untreated CAPS patients after stimulation for 48 h with phytohaemagglutinin (PHA), PHA plus IL-12, lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. We measured IL-1β, IL-6, IL-10, tumour necrosis factor (TNF), IL-12p70 and IFN-γ in the supernatants. PBMCs from three untreated CAPS patients were cultured in the presence of the IL-1β blocker Anakinra. Fifty healthy individuals served as controls. CAPS patients had high spontaneous production of IL-1β, IL-6, TNF and IFN-γ by unstimulated cells. However, stimulation indexes (SIs, ratio of stimulated to unstimulated production) of these cytokines to PHA and LPS were low in NOMID patients compared to controls. Unstimulated IL-10 and IL-12p70 production was normal, but up-regulation after PHA and LPS was also low. LPS plus IFN-γ inadequately up-regulated the production of IL-1β, IL-6, TNF and IL-10 in CAPS patients. In-vitro but not in-vivo treatment with Anakinra improved SIs by lowering spontaneous cytokine production. However, in-vitro treatment did not improve the low stimulated cytokine levels. Activating mutations in NLRP3 in CAPS are correlated with poor SIs to PHA, LPS and IFN-γ. The impairment in stimulated cytokine responses in spite of IL-1β blocking therapy suggests a broader intrinsic defect in CAPS patients, which is not corrected by targeting IL-1β. PMID:24773462

Impaired cytokine responses in patients with cryopyrin-associated periodic syndrome (CAPS).

PubMed

Haverkamp, M H; van de Vosse, E; Goldbach-Mansky, R; Holland, S M

2014-09-01

Cryopyrin-associated periodic syndrome (CAPS) is characterized by dysregulated inflammation with excessive interleukin (IL)-1β activation and secretion. Neonatal-onset multi-system inflammatory disease (NOMID) is the most severe form. We explored cytokine responses in 32 CAPS patients before and after IL-1β blocking therapy. We measured cytokines produced by activated peripheral blood monuclear cells (PBMCs) from treated and untreated CAPS patients after stimulation for 48 h with phytohaemagglutinin (PHA), PHA plus IL-12, lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. We measured IL-1β, IL-6, IL-10, tumour necrosis factor (TNF), IL-12p70 and IFN-γ in the supernatants. PBMCs from three untreated CAPS patients were cultured in the presence of the IL-1β blocker Anakinra. Fifty healthy individuals served as controls. CAPS patients had high spontaneous production of IL-1β, IL-6, TNF and IFN-γ by unstimulated cells. However, stimulation indexes (SIs, ratio of stimulated to unstimulated production) of these cytokines to PHA and LPS were low in NOMID patients compared to controls. Unstimulated IL-10 and IL-12p70 production was normal, but up-regulation after PHA and LPS was also low. LPS plus IFN-γ inadequately up-regulated the production of IL-1β, IL-6, TNF and IL-10 in CAPS patients. In-vitro but not in-vivo treatment with Anakinra improved SIs by lowering spontaneous cytokine production. However, in-vitro treatment did not improve the low stimulated cytokine levels. Activating mutations in NLRP3 in CAPS are correlated with poor SIs to PHA, LPS and IFN-γ. The impairment in stimulated cytokine responses in spite of IL-1β blocking therapy suggests a broader intrinsic defect in CAPS patients, which is not corrected by targeting IL-1β. © 2014 British Society for Immunology.

Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

PubMed

Zytek, Malgorzata; Kowalska, Joanna; Lukaszewicz, Maciej; Wojtczak, Blazej A; Zuberek, Joanna; Ferenc-Mrozek, Aleksandra; Darzynkiewicz, Edward; Niedzwiecka, Anna; Jemielity, Jacek

2014-12-07

A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

Primate translational vestibuloocular reflexes. I. High-frequency dynamics and three-dimensional properties during lateral motion

NASA Technical Reports Server (NTRS)

Angelaki, D. E.; McHenry, M. Q.; Hess, B. J.

2000-01-01

The dynamics and three-dimensional (3-D) properties of the primate translational vestibuloocular reflex (trVOR) for high-frequency (4-12 Hz, +/-0.3-0.4 g) lateral motion were investigated during near-target viewing at center and eccentric targets. Horizontal response gains increased with frequency and depended on target eccentricity. The larger the horizontal and vertical target eccentricity, the steeper the dependence of horizontal response gain on frequency. In addition to horizontal eye movements, robust torsional response components also were present at all frequencies. During center-target fixation, torsional response phase was opposite (anticompensatory) to that expected for an "apparent" tilt response. Instead torsional response components depended systematically on vertical-target eccentricity, increasing in amplitude when looking down and reversing phase when looking up. As a result the trVOR eye velocity vector systematically tilted away from a purely horizontal direction, through an angle that increased with vertical eccentricity with a slope of approximately 0.7. This systematic dependence of torsional eye velocity tilt on vertical eye position suggests that the trVOR might follow the 3-D kinematic requirements that have been shown to govern visually guided eye movements and near-target fixation.

Lexical Discourse Analysis in Translation

ERIC Educational Resources Information Center

Al Khotaba, Eissa; Al Tarawneh, Khaled

2015-01-01

Lexical Discourse very often depend on lexis. Lexical Discourse analysis, however, has not yet been given enough consideration of the phenomenon of translation. This paper investigates lexical discourse analysis in translation from one language to another. This qualitative study comprises 15 text translated by M.A. students at the Department of…

Genetics Home Reference: cap myopathy

MedlinePlus

... Email Facebook Twitter Home Health Conditions Cap myopathy Cap myopathy Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Cap myopathy is a disorder that primarily affects skeletal ...

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

PubMed

Fleith, Renata C; Mears, Harriet V; Leong, Xin Yun; Sanford, Thomas J; Emmott, Edward; Graham, Stephen C; Mansur, Daniel S; Sweeney, Trevor R

2018-06-01

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.

Targeting Tumor Microenvironment with Silibinin: Promise and Potential for a Translational Cancer Chemopreventive Strategy

PubMed Central

Deep, Gagan; Agarwal, Rajesh

2014-01-01

Tumor microenvironment (TME) refers to the dynamic cellular and extra-cellular components surrounding tumor cells at each stage of the carcinogenesis. TME has now emerged as an integral and inseparable part of the carcinogenesis that plays a critical role in tumor growth, angiogenesis, epithelial to mesenchymal transition (EMT), invasion, migration and metastasis. Besides its vital role in carcinogenesis, TME is also a better drug target because of its relative genetic stability with lesser probability for the development of drug-resistance. Several drugs targeting the TME (endothelial cells, macrophages, cancer-associated fibroblasts, or extra-cellular matrix) have either been approved or are in clinical trials. Recently, non-steroidal anti-inflammatory drugs targeting inflammation were reported to also prevent several cancers. These exciting developments suggest that cancer chemopreventive strategies targeting both tumor and TME would be better and effective towards preventing, retarding or reversing the process of carcinogenesis. Here, we have reviewed the effect of a well established hepatoprotective and chemopreventive agent silibinin on cellular (endothelial, fibroblast and immune cells) and non-cellular components (cytokines, growth factors, proteinases etc.) of the TME. Silibinin targets TME constituents as well as their interaction with cancer cells, thereby inhibiting tumor growth, angiogenesis, inflammation, EMT, and metastasis. Silibinin is already in clinical trials, and based upon completed studies we suggest that its chemopreventive effectiveness should be verified through its effect on biological end points in both tumor and TME. Overall, we believe that the chemopreventive strategies targeting both tumor and TME have practical and translational utility in lowering the cancer burden. PMID:23617249

Pharmacodynamic target attainment analysis against Streptococcus pneumoniae using levofloxacin 500 mg, 750 mg and 1000 mg once daily in plasma (P) and epithelial lining fluid (ELF) of hospitalized patients with community acquired pneumonia (CAP).

PubMed

Noreddin, Ayman M; Marras, Theodore K; Sanders, Kevin; Chan, Charles K N; Hoban, Daryl J; Zhanel, George G

2004-11-01

The pharmacokinetics and pharmacodynamics of levofloxacin in patients with respiratory infections such as community-acquired pneumonia (CAP) are poorly documented. This work aimed at assessing the pharmacodynamic target attainment against Streptococcus pneumoniae using levofloxacin 500 mg, 750 mg and 1000 mg administered once daily in plasma (P) and epithelial lining fluid (ELF) of hospitalized patients with community acquired pneumonia. The pharmacokinetics of levofloxacin in elderly (>/=65 years) compared with younger patients (<65 years) hospitalized with CAP were simulated. Susceptibility data with S. pneumoniae from our ongoing national surveillance study (Canadian Respiratory Organism Susceptibility Study-CROSS) were then used to produce pharmacodynamic indices of AUC(0-24)/MIC(all.) Monte Carlo simulations were then used to analyse target attainment of levofloxacin using doses of 500 mg, 750 mg and 1000 mg once daily to achieve free drug AUC(0-24)/MIC(all) >/= 30-100 versus S. pneumoniae in patients with CAP. Pharmacokinetics of levofloxacin simulated after 500 mg, 750 mg and 1000 mg once daily dosing resulted in levofloxacin volume of distribution: elderly patients = younger patients, while levofloxacin clearance was: elderly patients < younger patients. Levofloxacin t(1/2) values were longer in elderly patients (9.8 +/- 2.5h) than younger patients with CAP (7.4 +/- 2.5h). Free levofloxacin AUC(0-24) as well as AUC(0-24)/MIC(all) for S. pneumoniae were higher in elderly patients than younger patients. Monte Carlo simulation using levofloxacin 500 mg yielded probabilities of achieving free-drug AUC(0-24)/MIC(all) of 30 in P and ELF (95.7% and 98.1%) in elderly and younger patients (72.7% and 80.6%) respectively. Levofloxacin 750 mg and 1000 mg once daily had probability of achieving free-drug AUC(0-24)/MIC(all) of 30 in P/ELF of 98.1%/98.6% and 99.2%/99.0%, respectively, in elderly patients compared with 89.9%/94.1% and 95.2%/96.5%, respectively, for younger

AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

PubMed

Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

2015-10-01

Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

The rebirth of the cervical cap.

PubMed

Cappiello, J D; Grainger-Harrison, M

1981-01-01

In an effort to dispel myths surrounding the cervical cap, the historical and political factors affecting the cap's use in the U.S. are described. Clinical aspects of cap fitting are also included. The cervical cap has found only limited acceptance in the U.S. Skepticisms on the part of physicians may be the result of 2 factors: confusion of the cervical cap with intracervical devices used for artificial insemination and confusion with stem pessaries; and the lack of clinical research and statistical evaluation of efficacy rates. The latter factor prompted Tietze et al. to conduct the only U.S. statistical study of the cap in 1953. Of the 143 women studied, the pregnancy rate was 7.6/100 years of use. Of the 28 unplanned pregnancies, 6 were related to faulty technique or omission of a spermicide and 10 were instances of admittedly irregular use. When these failures are omitted, the theoretical effectiveness rate is about 98%. Some practitioners are concerned about an increased incidence of cervical erosion with cap use. Possibly currently conducted studies will show that cap and spermicide users have a lower incidence of cervical erosion than women using no contraceptive method. Study findings suggest that the cervical cap may afford protection without any spermicidal supplement, but the use of spermicides continues to be recommended to clients. Advantages of the cervical cap include the following: it can be left in place longer than a diaphragm without additional applications of spermicide in the vagina; and the insertion of the cap is unrelated to the time of intercourse. Despite research on toleration of the cap for 3 weeks at a time, it is recommended that the cap be worn for only a few days at a time. At this time there are no manufacturers of cervical caps for contraceptive use in the U.S. The cap is now being imported from England and it costs $6.00. A factor that has made the cap unpopular with many physicians is the lengthy time required for fitting. An

High-pressure jet cutters improve capping operations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abel, L.W.; Campbell, P.J.; Bowden, J.R. Sr.

1995-05-08

Advances in abrasive cutting technology have improved the methods for removing damaged equipment and preparing wellheads for capping. This technology, much of which was refined during well control operations in Kuwait in 1991, can improve the safety and efficiency of capping jobs by cutting wellheads or casing quickly and cleanly. The majority of well control jobs involve one of three types of capping operations: capping to a flange, capping by installing a wellhead, or capping to a casing stub. Capping operations are often the first major step in regaining control of the well during blowout intervention. Proper planning of amore » capping operation must take into account the mass flow rate, combustible nature of the flow, well bore geometry, and operations in the post-capping phase of the project. The paper discusses capping vehicles, tree removal, jet cutters, capping to a flange, capping to a stub, swallowing the stub, spin-on technique, capping on fire, stinging, offshore blowouts, firefighting, pollution control, intervention equipment, and rig removal.« less

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

PubMed

Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

2016-05-21

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

TFIIH and P-TEFb Coordinate Transcription with Capping Enzyme Recruitment at Specific Genes in Fission Yeast

PubMed Central

Viladevall, Laia; St. Amour, Courtney V.; Rosebrock, Adam; Schneider, Susanne; Zhang, Chao; Allen, Jasmina J.; Shokat, Kevan M.; Schwer, Beate; Leatherwood, Janet K.; Fisher, Robert P.

2009-01-01

Summary Cyclin-dependent kinases (CDKs) are subunits of transcription factor (TF) IIH and positive transcription elongation factor b (P-TEFb). To define their functions, we mutated the TFIIH-associated kinase Mcs6 and P-TEFb homologs Cdk9 and Lsk1 of fission yeast, making them sensitive to bulky purine analogs. Selective inhibition of Mcs6 or Cdk9 blocks cell division, alters RNA polymerase (Pol) II carboxyl-terminal domain (CTD) phosphorylation and represses specific, overlapping subsets of transcripts. At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5—a CDK substrate and regulator of elongation—accumulates disproportionately to Pol II when either kinase is inhibited. In contrast, Mcs6 activity is sufficient, and necessary, to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs. PMID:19328067

Redox Control and Hydrogen Production in Sediment Caps Using Carbon Cloth Electrodes

PubMed Central

Sun, Mei; Yan, Fei; Zhang, Ruiling; Reible, Danny D.; Lowry, Gregory V.; Gregory, Kelvin B.

2010-01-01

Sediment caps that degrade contaminants can improve their ability to contain contaminants relative to sand and sorbent-amended caps, but few methods to enhance contaminant degradation in sediment caps are available. The objective of this study was to determine if, carbon electrodes emplaced within a sediment cap at poised potential could create a redox gradient and provide electron donor for the potential degradation of contaminants. In a simulated sediment cap overlying sediment from the Anacostia River (Washington, DC), electrochemically induced redox gradients were developed within 3 days and maintained over the period of the test (~100 days). Hydrogen and oxygen were produced by water electrolysis at the electrode surfaces and may serve as electron donor and acceptor for contaminant degradation. Electrochemical and geochemical factors that may influence hydrogen production were studied. Hydrogen production displayed zero order kinetics with ~75% coulombic efficiency and rates were proportional to the applied potential between 2.5V to 5V and not greatly affected by pH. Hydrogen production was promoted by increasing ionic strength and in the presence of natural organic matter. Graphite electrode-stimulated degradation of tetrachlorobenzene in a batch reactor was dependent on applied voltage and production of hydrogen to a concentration above the threshold for biological dechlorination. These findings suggest that electrochemical reactive capping can potentially be used to create “reactive” sediments caps capable of promoting chemical or biological transformations of contaminants within the cap. PMID:20879761

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

PubMed Central

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

PubMed

Gavilan, Maria P; Arjona, Marina; Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R; Bornens, Michel; Rios, Rosa M

2015-03-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

PubMed Central

Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

2015-01-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

Comparative ecotoxicity of chlorantraniliprole to non-target soil invertebrates.

PubMed

Lavtižar, Vesna; Berggren, Kristina; Trebše, Polonca; Kraak, Michiel H S; Verweij, Rudo A; van Gestel, Cornelis A M

2016-09-01

The insecticide chlorantraniliprole (CAP) is gaining importance in agricultural practice, but data on its possible negative effects on non-target organisms is severely deficient. This study therefore determined CAP toxicity to non-target soil invertebrates playing a crucial role in ecosystem functioning, including springtails (Folsomia candida), isopods (Porcellio scaber), enchytraeids (Enchytraeus crypticus) and oribatid mites (Oppia nitens). In sublethal toxicity tests in Lufa 2.2 soil, chronic exposure to CAP concentrations up to 1000 mg/kgdw did not affect the survival and reproduction of E. crypticus and O. nitens nor the survival, body weight and consumption of P. scaber. In contrast, the survival and reproduction of F. candida was severely affected, with an EC50 for effects on reproduction of 0.14 mg CAP/kgdw. The toxicity of CAP to the reproduction of F. candida was tested in four different soils following OECD guideline 232, and additionally in an avoidance test according to ISO guideline 17512-2. A significantly lower toxicity in soils rich in organic matter was observed, compared to low organic soils. Observations in the avoidance test with F. candida suggest that CAP acted in a prompt way, by affecting collembolan locomotor abilities thus preventing them from escaping contaminated soil. This study shows that CAP may especially pose a risk to non-target soil arthropods closely related to insects, while other soil invertebrates seem rather insensitive. Copyright © 2016 Elsevier Ltd. All rights reserved.

North Polar Cap

NASA Technical Reports Server (NTRS)

2004-01-01

7 September 2004 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a 1.4 m/pixel (5 ft/pixel) view of a typical martian north polar ice cap texture. The surface is pitted and rough at the scale of several meters. The north polar residual cap of Mars consists mainly of water ice, while the south polar residual cap is mostly carbon dioxide. This picture is located near 85.2oN, 283.2oW. The image covers an area approximately 1 km wide by 1.4 km high (0.62 by 0.87 miles). Sunlight illuminates this scene from the lower left.

Synthesis and evaluation of hetero- and homo-dimers of ribosome-targeting antibiotics: Antimicrobial activity, in vitro inhibition of translation, and drug resistance

PubMed Central

Berkov-Zrihen, Yifat; Green, Keith D.; Labby, Kristin J.; Feldman, Mark; Garneau-Tsodikova, Sylvie; Fridman, Micha

2013-01-01

In this study, we describe the synthesis of a full set of homo- and hetero-dimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived. PMID:23786357

Results and Outlook of The Aluminum Capture Experiment (AlCap)

NASA Astrophysics Data System (ADS)

Quirk, John R.; Miller, James; ALCap Collaboration Collaboration

2016-03-01

Observation of neutrinoless muon-to-electron conversion in the presence of a nucleus would be unambiguous evidence of physics Beyond the Standard Model. Two experiments, COMET at J-PARC and Mu2e at Fermilab, will search for this process in the coming decade. Barring discovery, these experiments will provide upper-limits on this branching ratio up to 10,000 times better than previously published. COMET/Mu2e developed a joint venture, the AlCap Experiment, to measure particle emission spectra from muonic interactions in a number of materials. As a major source of background hits in COMET/Mu2e detectors, AlCap sought to measure the charged particle and neutron spectra following nuclear capture on the candidate target materials aluminum and titanium. Additionally, COMET/Mu2e are exploring normalization schemes via AlCap's measurement of the photon spectra following both atomic and nuclear capture. Over the course of 2013 and 2015, AlCap performed three runs at the Paul Scherrer Institut in Switzerland. The first acquired preliminary data for all spectra, the second run collected only neutron and photon data, and the third primarily charged particle data. Preliminary analyses of the first two runs, already impactful for COMET/Mu2e, is presented along with a summary of the third.

75 FR 49527 - Caps Visual Communications, LLC; Black Dot Group; Formerly Known as Caps Group Acquisition, LLC...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-13

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,195] Caps Visual Communications, LLC; Black Dot Group; Formerly Known as Caps Group Acquisition, LLC Chicago, IL; Amended... of Caps Visual Communications, LLC, Black Dot Group, formerly known as Caps Group Acquisition, LLC...

Influence of rate of force application during compression on tablet capping.

PubMed

Sarkar, Srimanta; Ooi, Shing Ming; Liew, Celine Valeria; Heng, Paul Wan Sia

2015-04-01

Root cause and possible processing remediation of tablet capping were investigated using a specially designed tablet press with an air compensator installed above the precompression roll to limit compression force and allow extended dwell time in the precompression event. Using acetaminophen-starch (77.9:22.1) as a model formulation, tablets were prepared by various combinations of precompression and main compression forces, set precompression thickness, and turret speed. The rate of force application (RFA) was the main factor contributing to the tablet mechanical strength and capping. When target force above the force required for strong interparticulate bond formation, the resultant high RFA contributed to more pronounced air entrapment, uneven force distribution, and consequently, stratified densification in compact together with high viscoelastic recovery. These factors collectively had contributed to the tablet capping. As extended dwell time assisted particle rearrangement and air escape, a denser and more homogenous packing in the die could be achieved. This occurred during the extended dwell time when a low precompression force was applied, followed by application of main compression force for strong interparticulate bond formation that was the most beneficial option to solve capping problem. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

A front-end wafer-level microsystem packaging technique with micro-cap array

NASA Astrophysics Data System (ADS)

Chiang, Yuh-Min

2002-09-01

The back-end packaging process is the remaining challenge for the micromachining industry to commercialize microsystem technology (MST) devices at low cost. This dissertation presents a novel wafer level protection technique as a final step of the front-end fabrication process for MSTs. It facilitates improved manufacturing throughput and automation in package assembly, wafer level testing of devices, and enhanced device performance. The method involves the use of a wafer-sized micro-cap array, which consists of an assortment of small caps micro-molded onto a material with adjustable shapes and sizes to serve as protective structures against the hostile environments during packaging. The micro-cap array is first constructed by a micromachining process with micro-molding technique, then sealed to the device wafer at wafer level. Epoxy-based wafer-level micro cap array has been successfully fabricated and showed good compatibility with conventional back-end packaging processes. An adhesive transfer technique was demonstrated to seal the micro cap array with a MEMS device wafer. No damage or gross leak was observed while wafer dicing or later during a gross leak test. Applications of the micro cap array are demonstrated on MEMS, microactuators fabricated using CRONOS MUMPS process. Depending on the application needs, the micro-molded cap can be designed and modified to facilitate additional component functions, such as optical, electrical, mechanical, and chemical functions, which are not easily achieved in the device by traditional means. Successful fabrication of a micro cap array comprised with microlenses can provide active functions as well as passive protection. An optical tweezer array could be one possibility for applications of a micro cap with microlenses. The micro cap itself could serve as micro well for DNA or bacteria amplification as well.

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development

PubMed Central

Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

2016-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants. PMID:28358146

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development.

PubMed

Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

2016-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants.

Lost in Translation: Assessing Effectiveness of Focus Group Questioning Techniques to Develop Improved Translation of Terminology Used in HIV Prevention Clinical Trials

PubMed Central

Mack, Natasha; Ramirez, Catalina B.; Friedland, Barbara; Nnko, Soori

2013-01-01

Introduction Achieving participant comprehension has proven to be one of the most difficult, practical, and ethical challenges of HIV prevention clinical trials. It becomes even more challenging when local languages do not have equivalent scientific and technical vocabularies, rendering communication of scientific concepts in translated documents extremely difficult. Even when bilingual lexicons are developed, there is no guarantee that participants understand the terminology as translated. Methods We conducted twelve focus groups with women of reproductive age in Mwanza, Tanzania to explore the effectiveness of four questioning techniques for: (1) assessing participants' familiarity with existing technical terms and concepts, (2) generating a list of acceptable technical and non-technical terms, (3) testing our definitions of technical terms, and (4) verifying participants' preferences for terms. Focus groups were transcribed, translated, and qualitatively analyzed. Results and Discussion A translation process that uses all four questioning techniques in a step-wise approach is an effective way to establish a baseline understanding of participants' familiarity with research terms, to develop and test translatable definitions, and to identify participants' preferred terminology for international HIV clinical research. This may help to ensure that important concepts are not “lost in translation.” The results emphasize the importance of using a variety of techniques depending on the level of participant familiarity with research concepts, the existence of colloquial or technical terms in the target language, and the inherent complexity of the terms. PMID:24040075

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

PubMed

Oem, Jae-Ku; Xiang, Zhonghua; Zhou, Yan; Babiuk, Lorne A; Liu, Qiang

2007-09-01

Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.

Size-confined fixed-composition and composition-dependent engineered band gap alloying induces different internal structures in L-cysteine-capped alloyed quaternary CdZnTeS quantum dots

NASA Astrophysics Data System (ADS)

Adegoke, Oluwasesan; Park, Enoch Y.

2016-06-01

The development of alloyed quantum dot (QD) nanocrystals with attractive optical properties for a wide array of chemical and biological applications is a growing research field. In this work, size-tunable engineered band gap composition-dependent alloying and fixed-composition alloying were employed to fabricate new L-cysteine-capped alloyed quaternary CdZnTeS QDs exhibiting different internal structures. Lattice parameters simulated based on powder X-ray diffraction (PXRD) revealed the internal structure of the composition-dependent alloyed CdxZnyTeS QDs to have a gradient nature, whereas the fixed-composition alloyed QDs exhibited a homogenous internal structure. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis confirmed the size-confined nature and monodispersity of the alloyed nanocrystals. The zeta potential values were within the accepted range of colloidal stability. Circular dichroism (CD) analysis showed that the surface-capped L-cysteine ligand induced electronic and conformational chiroptical changes in the alloyed nanocrystals. The photoluminescence (PL) quantum yield (QY) values of the gradient alloyed QDs were 27-61%, whereas for the homogenous alloyed QDs, the PL QY values were spectacularly high (72-93%). Our work demonstrates that engineered fixed alloying produces homogenous QD nanocrystals with higher PL QY than composition-dependent alloying.

Effect of high-latitude ionospheric convection on Sun-aligned polar caps

NASA Technical Reports Server (NTRS)

Sojka, J. J.; Zhu, L.; Crain, D. J.; Schunk, R. W.

1994-01-01

A coupled magnetospheric-ionospheric (M-I) magnetohydrodynamic (MHD) model has been used to simulate the formation of Sun-aligned polar cap arcs for a variety of interplanetary magnetic field (IMF) dependent polar cap convection fields. The formation process involves launching an Alfven shear wave from the magnetosphere to the ionosphere where the ionospheric conductance can react self-consistently to changes in the upward currents. We assume that the initial Alfven shear wave is the result of solar wind-magnetosphere interactions. The simulations show how the E region density is affected by the changes in the electron precipitation that are associated with the upward currents. These changes in conductance lead to both a modified Alfven wave reflection at the ionosphere and the generation of secondary Alfven waves in the ionosphere. The ensuing bouncing of the Alfven waves between the ionosphere and magnetosphere is followed until an asymptotic solution is obtained. At the magnetosphere the Alfven waves reflect at a fixed boundary. The coupled M-I Sun-aligned polar cap arc model of Zhu et al.(1993a) is used to carry out the simulations. This study focuses on the dependence of the polar cap arc formation on the background (global) convection pattern. Since the polar cap arcs occur for northward and strong B(sub y) IMF conditions, a variety of background convection patterns can exist when the arcs are present. The study shows that polar cap arcs can be formed for all these convection patterns; however, the arc features are dramatically different for the different patterns. For weak sunward convection a relatively confined single pair of current sheets is associated with the imposed Alfven shear wave structure. However, when the electric field exceeds a threshold, the arc structure intensifies, and the conductance increases as does the local Joule heating rate. These increases are faster than a linear dependence on the background electric field strength. Furthermore

nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

PubMed Central

Gandin, Valentina; Masvidal, Laia; Hulea, Laura; Gravel, Simon-Pierre; Cargnello, Marie; McLaughlan, Shannon; Cai, Yutian; Balanathan, Preetika; Morita, Masahiro; Rajakumar, Arjuna; Furic, Luc; Pollak, Michael; Porco, John A.; St-Pierre, Julie; Pelletier, Jerry; Larsson, Ola; Topisirovic, Ivan

2016-01-01

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5′ TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5′ TOP motif but that 5′ UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5′ UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5′ UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5′ UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5′ UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5′ UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells. PMID:26984228

Evaluation of a modified Fitts law brain-computer interface target acquisition task in able and motor disabled individuals

NASA Astrophysics Data System (ADS)

Felton, E. A.; Radwin, R. G.; Wilson, J. A.; Williams, J. C.

2009-10-01

A brain-computer interface (BCI) is a communication system that takes recorded brain signals and translates them into real-time actions, in this case movement of a cursor on a computer screen. This work applied Fitts' law to the evaluation of performance on a target acquisition task during sensorimotor rhythm-based BCI training. Fitts' law, which has been used as a predictor of movement time in studies of human movement, was used here to determine the information transfer rate, which was based on target acquisition time and target difficulty. The information transfer rate was used to make comparisons between control modalities and subject groups on the same task. Data were analyzed from eight able-bodied and five motor disabled participants who wore an electrode cap that recorded and translated their electroencephalogram (EEG) signals into computer cursor movements. Direct comparisons were made between able-bodied and disabled subjects, and between EEG and joystick cursor control in able-bodied subjects. Fitts' law aptly described the relationship between movement time and index of difficulty for each task movement direction when evaluated separately and averaged together. This study showed that Fitts' law can be successfully applied to computer cursor movement controlled by neural signals.

RNA-dependent RNA polymerase: Addressing Zika outbreak by a phylogeny-based drug target study.

PubMed

Stephen, Preyesh; Lin, Sheng-Xiang

2018-01-01

Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. © 2017 John Wiley & Sons A/S.

North Polar Cap

NASA Technical Reports Server (NTRS)

2004-01-01

[figure removed for brevity, see original site]

This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.

In the images of the Martian north polar cap we can see these same type of winds. Notice the streamers of dust moving downslope over the darker trough sides, these streamers show the laminar flow regime coming off the cap. Within the trough we see turbulent clouds of dust, kicked up at the trough base as the winds slow down and enter a chaotic flow regime.

The horizontal lines in these images are due to framelet overlap and lighting conditions over the bright polar cap.

Image information: VIS instrument. Latitude 86.5, Longitude 64.5 East (295.5 West). 40 meter/pixel resolution.

Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation

North Polar Cap

NASA Technical Reports Server (NTRS)

2004-01-01

[figure removed for brevity, see original site]

This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.