212 research outputs found

    Protein interactions in Xenopus germ plasm RNP particles

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    Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles

    Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

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    We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

    Helicobacter Pylori Infection in Pediatric Patients Living in Europe: Results of the EuroPedHP Registry 2013 to 2016

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    Objectives: The aim of the study was to assess clinical presentation, endoscopic findings, antibiotic susceptibility and treatment success of Helicobacter pylori (H. pylori) infected pediatric patients. Methods: Between 2013 and 2016, 23 pediatric hospitals from 17 countries prospectively submitted data on consecutive H. pylori-infected (culture positive) patients to the EuroPedHP-Registry. Results: Of 1333 patients recruited (55.1% girls, median age 12.6 years), 1168 (87.6%) were therapy naïve (group A) and 165 (12.4%) had failed treatment (group B). Patients resided in North/Western (29.6%), Southern (34.1%) and Eastern Europe (23.0%), or Israel/Turkey (13.4%). Main indications for endoscopy were abdominal pain or dyspepsia (81.2%, 1078/1328). Antral nodularity was reported in 77.8% (1031/1326) of patients, gastric or duodenal ulcers and erosions in 5.1% and 12.8%, respectively. Primary resistance to clarithromycin (CLA) and metronidazole (MET) occurred in 25% and 21%, respectively, and increased after failed therapy. Bacterial strains were fully susceptible in 60.5% of group A, but in only 27.4% of group B. Primary CLA resistance was higher in Southern and Eastern Europe (adjusted odds ratio [ORadj] = 3.44, 95% confidence interval [CI] 2.22-5.32, P < 0.001 and 2.62, 95% CI: 1.63-4.22, P < 0.001, respectively) compared with Northern/Western Europe. Children born outside Europe showed higher primary MET resistance (ORadj = 3.81, 95% CI: 2.25-6.45, P < 0.001). Treatment success in group A reached only 79.8% (568/712) with 7 to 14 days triple therapy tailored to antibiotic susceptibility. Conclusions: Peptic ulcers are rare in dyspeptic H. pylori-infected children. Primary resistance to CLA and MET is markedly dependent on geographical regions of birth and residence. The ongoing survey will show whether implementation of the updated ESPGHAN/NASPGHAN guidelines will improve the eradication success.info:eu-repo/semantics/publishedVersio

    Low prevalence of H. pylori Infection in HIV-Positive Patients in the Northeast of Brazil

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    <p>Abstract</p> <p>Background</p> <p>This study conducted in Northeastern Brazil, evaluated the prevalence of <it>H. pylori </it>infection and the presence of gastritis in HIV-infected patients.</p> <p>Methods</p> <p>There were included 113 HIV-positive and 141 age-matched HIV-negative patients, who underwent upper gastrointestinal endoscopy for dyspeptic symptoms. <it>H. pylori </it>status was evaluated by urease test and histology.</p> <p>Results</p> <p>The prevalence of <it>H. pylori </it>infection was significantly lower (p < 0.001) in HIV-infected (37.2%) than in uninfected (75.2%) patients. There were no significant differences between <it>H. pylori </it>status and gender, age, HIV viral load, antiretroviral therapy and the use of antibiotics. A lower prevalence of <it>H. pylori </it>was observed among patients with T CD4 cell count below 200/mm<sup>3</sup>; however, it was not significant. Chronic active antral gastritis was observed in 87.6% of the HIV-infected patients and in 780.4% of the control group (p = 0.11). <it>H. pylori </it>infection was significantly associated with chronic active gastritis in the antrum in both groups, but it was not associated with corpus chronic active gastritis in the HIV-infected patients.</p> <p>Conclusion</p> <p>We demonstrated that the prevalence of <it>H. pylori </it>was significantly lower in HIV-positive patients compared with HIV-negative ones. However, corpus gastritis was frequently observed in the HIV-positive patients, pointing to different mechanisms than <it>H. pylori </it>infection in the genesis of the lesion.</p

    Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

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    Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

    Solution Structure of Kurtoxin: A Gating Modifier Selective for Cav3 Voltage-Gated Ca2+ Channels

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    Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms

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    Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses
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