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Find video protocols related to scientific articles indexed in Pubmed.
L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES.
FEBS Lett.
PUBLISHED: 07-28-2014
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The leader protease (L(pro)) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L(pro) is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L(pro), significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).
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Subtypes of Batterers in Treatment: Empirical Support for a Distinction between Type I, Type II and Type III.
PLoS ONE
PUBLISHED: 01-01-2014
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This study explores the existence of different types of batterers in a sample of 266 men who had been court referred for intimate partner violence. The data collected in the assessment that have been used to perform a hierarchical and a two-step cluster analysis fall into three areas: aggression towards the partner, general aggression and presence of psychopathology and personality traits, more specifically, alcohol use, borderline and antisocial personality traits, psychopathy traits, state anger and trait anger, anger expression and control, anger, hostility, and, finally, impulsivity. The results show a typology consisting of 3 types of batterers on the basis of violence level and psychopathology: low (65%), moderate (27.8%) and high (7.1%). This study provides empirical support for the development of batterer typologies. These typologies will help achieve early detection of different types of batterers, allowing us to tailor interventions on the basis of the needs of each of the types.
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Translation of viral mRNAs that do not require eIF4E is blocked by the inhibitor 4EGI-1.
Virology
PUBLISHED: 03-23-2013
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High throughput screening has rendered new inhibitors of eukaryotic protein synthesis. One such molecule, 4EGI-1 has been reported to selectively block the initiation factor eIF4E. We have investigated the action of this inhibitor on translation directed by several viral mRNAs which, in principle, do not utilize eIF4E. We found that 4EGI-1 inhibits translation directed by poliovirus IRES, in rabbit reticulocyte lysates, to a similar extent as capped mRNA. Moreover, 4EGI-1 inhibits translation driven by poliovirus IRES, both in vitro and in cultured cells, despite cleavage of eIF4G by picornavirus proteases. Finally, translation of vesicular stomatitis virus mRNAs and Sindbis virus subgenomic mRNA is blocked by 4EGI-1 in infected cells to a similar extent as cellular mRNAs. These findings cast doubt on the selective action of this inhibitor, and suggest that this molecule may affect other steps in protein synthesis unrelated to cap recognition by eIF4E.
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Membrane integration of poliovirus 2B viroporin.
J. Virol.
PUBLISHED: 08-10-2011
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Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.
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Translation without eIF2 promoted by poliovirus 2A protease.
PLoS ONE
PUBLISHED: 07-05-2011
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Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2A(pro) alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2A(pro) variant unable to cleave eIF4G. The level of 2A(pro) synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2A(pro) at high concentrations is necessary. The finding that 2A(pro) provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis.
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Translation of viral mRNA without active eIF2: the case of picornaviruses.
PLoS ONE
PUBLISHED: 03-18-2011
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Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2? for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2? whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2? by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2? also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2?-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2?, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2? when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.
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Translation driven by picornavirus IRES is hampered from Sindbis virus replicons: rescue by poliovirus 2A protease.
J. Mol. Biol.
PUBLISHED: 05-05-2010
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Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A(pro)) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A(pro) variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A(pro) or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A(pro). These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.
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Cell permeabilization by poliovirus 2B viroporin triggers bystander permeabilization in neighbouring cells through a mechanism involving gap junctions.
Cell. Microbiol.
PUBLISHED: 03-12-2010
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Poliovirus 2B protein is a well-known viroporin implicated in plasma membrane permeabilization to ions and low-molecular-weight compounds during infection. Translation in mammalian cells expressing 2B protein is inhibited by hygromycin B (HB) but remains unaffected in mock cells, which are not permeable to the inhibitor. Here we describe a previously unreported bystander effect in which healthy baby hamster kidney (BHK) cells become sensitive to HB when co-cultured with a low proportion of cells expressing poliovirus 2B. Viroporins E from mouse hepatitis virus, 6K from Sindbis virus and NS4A protein from hepatitis C virus were also able to permeabilize neighbouring cells to different extents. Expression of 2B induced permeabilization of neighbouring cell lines other than BHK. We found that gap junctions are responsible mediating the observed bystander permeabilization. Gap junctional communication was confirmed in 2B-expressing co-cultures by fluorescent dye transfer. Moreover, the presence of connexin 43 was confirmed in both mock and 2B-transfected cells. Finally, inhibition of HB entry to neighbouring cells was observed with 18alpha-glycyrrhethinic acid, an inhibitor of gap junctions. Taken together, these findings support a mechanism involving gap junctional intercellular communication in the bystander permeabilization effect observed in healthy cells co-cultured with poliovirus 2B-expressing cells.
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Immunohistochemical detection of intravascular platelet microthrombi in patients with lupus nephritis and anti-phospholipid antibodies.
Rheumatology (Oxford)
PUBLISHED: 06-19-2009
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To evaluate whether the use of platelet immunohistochemistry (IHC) markers improves the sensitivity of histological methods to detect microthrombosis in SLE nephritis and aPLs and to analyse the clinicopathological correlations of microthrombosis in this setting.
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Translation directed by hepatitis A virus IRES in the absence of active eIF4F complex and eIF2.
PLoS ONE
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Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A(pro) or L(pro). The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A(pro) strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L(pro) strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A(pro) and L(pro) exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L(pro). Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2? has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.
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Phosphorylation of eIF2? is responsible for the failure of the picornavirus internal ribosome entry site to direct translation from Sindbis virus replicons.
J. Gen. Virol.
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Translation directed by the poliovirus (PV) or encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) is very inefficient when expressed from Sindbis virus (SV) replicons. This inhibition can be rescued by co-expression of PV 2A protease (2A(pro)). Inhibition correlates with the extensive phosphorylation of eukaryotic initiation factor (eIF) 2? induced by SV replication. Confirmation that PV or EMCV IRES-driven translation can function when eIF2? is not phosphorylated was obtained in dsRNA-activated protein kinase knockout mouse embryonic fibroblasts (PKR(-/-) MEFs), where SV replication cannot induce eIF2? phosphorylation, and in variant S51A MEFs that express an unphosphorylatable eIF2?. In these cells, PV or EMCV IRES-dependent translation operated more efficiently than in wild-type MEFs. However, this translation was potently blocked when eIF2? was phosphorylated by the addition of thapsigargin to PKR(-/-) MEFs. In addition, when wild-type eIF2? was expressed in S51A MEFs or PKR was expressed in PKR(-/-) MEFs, PV IRES-dependent translation decreased. In both cases, the decrease in PV IRES-dependent translation correlated with the phosphorylation of eIF2?. Notably, PV 2A(pro) expression rescued PV IRES-driven translation in thapsigargin-treated PKR(-/-) MEFs. Taken together, these results demonstrated that PV IRES-driven translation can take place from SV replicons if eIF2? remains unphosphorylated. Remarkably, PV IRES-dependent translation was fully functional in this system when PV 2A(pro) was present, even if eIF2? was phosphorylated.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.