Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.     

Internet-Based Self-Help Intervention for ICD-11 Adjustment Disorder: Preliminary Findings

Adjustment disorder is one of the most diagnosed mental disorders. However, there is a lack of studies of specialized internet-based psychosocial interventions for adjustment disorder. We aimed to analyze the outcomes of an internet-based unguided self-help psychosocial intervention BADI for adjustment disorder in a two armed randomized controlled trial with a waiting list control group. In total 284 adult participants were randomized in this study. We measured adjustment disorder as a primary outcome, and psychological well-being as a secondary outcome at pre-intervention (T1) and one month after the intervention (T2). We found medium effect size of the intervention for the completer sample on adjustment disorder symptoms. Intervention was effective for those participants who used it at least one time in 30-day period. Our results revealed the potential of unguided internet-based self-help intervention for adjustment disorder. However, high dropout rates in the study limits the generalization of the outcomes of the intervention only to completers.     

Swedish and Lithuanian Students' Experience of Their Rights

One important implication of the UN Convention on the Rights of the Child is that every child should be part of democratic society. In the present empirical study, students' (aged 12–14, 16–18) conceptions of democracy in two Swedish and two Lithuanian schools are compared. Comparisons on age and gender are also examined. The analysis shows certain similarities in the Sweden and Lithuanian students' concept of democracy, but differences emerge.     

Targeting PBR by hexaminolevulinate-mediated photodynamic therapy induces apoptosis through translocation of apoptosis-inducing factor in human leukemia cells

Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.     

The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication

Two of the five viral-coded proteins of tombusviruses, which are small, nonsegmented, plus-stranded RNA viruses of plants, are required for replication in infected cells. These replicase proteins, namely, p33 and p92, of cucumber necrosis virus are expressed directly from the genomic RNA via a readthrough mechanism. Their overlapping domains contain an arginine/proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). Site-directed mutagenesis of p33 expressed in Escherichia coli, followed by a gel shift assay, defined two of the four arginines as required for efficient RNA binding in vitro. In vivo testing of 19 RPR motif mutants revealed that the RPR motif, and therefore the ability to bind RNA, is important for the replication of tombusviruses and their associated defective interfering (DI) RNAs. Mutation within the RPR motif also affected the ratio of subgenomic versus genomic RNAs in infected cells. To test whether the RPR motif is essential for the function of either p33 or p92 in replication, we used a two-component system developed by, J. Virol. 5845-5851), in which p92 was expressed from the genomic RNA of a tombusvirus, while p33 was expressed from a DI RNA. The protoplast experiments with the two-component system revealed that the RPR motif is essential for the replication function of both proteins. Interestingly, mutations within the RPR motif of p33 and p92 had different effects on RNA replication, suggesting different roles for the RNA-binding motifs of these proteins in tombusvirus replication.     

Impact of Eliminating Postprocedural Antibiotic Prophylaxis in Patients without Biliary Instrumentation or Bypass Undergoing Hepatic Artery Embolization for Hepatic Malignancies

PurposeTo assess the infection rate after eliminating postprocedural antibiotics in patients undergoing hepatic artery embolization (HAE) for primary and secondary hepatic malignancies.Material and MethodsIn this historical cohort study, adults ≥18 years of age without prior biliary instrumentation or bypass who underwent HAE and received pre- and postprocedure antibiotic prophylaxis between September 1, 2014, and August 31, 2015, comprised group A, whereas similar patients receiving only preprocedure antibiotic prophylaxis between October 1, 2015, and September 30, 2016, comprised group B. Procedures conducted between September 1, 2015, and September 30, 2015, were excluded. The primary outcome was any infection occurring within 30 days of HAE.ResultsA total of 150 patients underwent 204 HAE procedures in group A, and 171 patients underwent 221 procedures in group B. Cefazolin given as a 1-g dose (or 2 grams if obese) was administered in 391 of 425 evaluable procedures (92%). Clindamycin plus gentamicin was prescribed in 34 patients (8%) who had severe penicillin allergy. There was significant improvement in adherence to the postprocedure antibiotic regimen, from 68% (138 of 204 procedures) to 98% (216 of 221 procedures) (P < .001) with elimination of postprocedure prophylaxis. There were no significant differences in 30-day infection rates (5 [3%] vs. 5 [2%]; P = .57), hospital readmissions (13 [6%] vs. 12 [5%]; P = .68), or all-cause mortality (3 [1%] vs. 3 [1%]; P = .62) between the 2 groups.ConclusionsElimination of postprocedural antibiotics after HAE did not lead to an increase in infectious complications. This finding supports the 2018 Society of Interventional Radiology recommendation for preprocedural prophylaxis only for HAE in the setting of an intact sphincter of Oddi.