Eukaryotic translational termination efficiency is influenced by the 3' nucleotides within the ribosomal mRNA channel.

When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding release factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the release factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.

HIV-1 and Human PEG10 Frameshift Elements Are Functionally Distinct and Distinguished by Novel Small Molecule Modulators.

Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using -1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 µM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same generic mechanism.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

Continuous positive airway pressure (CPAP) during the postoperative period for prevention of postoperative morbidity and mortality following major abdominal surgery.

BACKGROUND: Major abdominal surgery can be associated with a number of serious complications that may impair patient recovery. In particular, postoperative pulmonary complications (PPCs), including respiratory complications such as atelectasis and pneumonia, are a major contributor to postoperative morbidity and may even contribute to increased mortality. Continuous positive airway pressure (CPAP) is a type of therapy that uses a high-pressure gas source to deliver constant positive pressure to the airways throughout both inspiration and expiration. This approach is expected to prevent some pulmonary complications, thus reducing mortality. OBJECTIVES: To determine whether any difference can be found in the rate of mortality and adverse events following major abdominal surgery in patients treated postoperatively with CPAP versus standard care, which may include traditional oxygen delivery systems, physiotherapy and incentive spirometry. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) 2013, Issue 9; Ovid MEDLINE (1966 to 15 September 2013); EMBASE (1988 to 15 September 2013); Web of Science (to September 2013) and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) (to September 2013). SELECTION CRITERIA: We included all randomized controlled trials (RCTs) in which CPAP was compared with standard care for prevention of postoperative mortality and adverse events following major abdominal surgery. We included all adults (adults as defined by individual studies) of both sexes. The intervention of CPAP was applied during the postoperative period. We excluded studies in which participants had received PEEP during surgery. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies that met the selection criteria from all studies identified by the search strategy. Two review authors extracted the data and assessed risk of bias separately, using a data extraction form. Data entry into RevMan was performed by one review author and was checked by another for accuracy. We performed a limited meta-analysis and constructed a summary of findings table. MAIN RESULTS: We selected 10 studies for inclusion in the review from 5236 studies identified in the search. These 10 studies included a total of 709 participants. Risk of bias for the included studies was assessed as high in six studies and as unclear in four studies.Two RCTs reported all-cause mortality. Among 413 participants, there was no clear evidence of a difference in mortality between CPAP and control groups, and considerable heterogeneity between trials was noted (risk ratio (RR) 1.28, 95% confidence interval (CI) 0.35 to 4.66; I(2) = 75%).Six studies reported demonstrable atelectasis in the study population. A reduction in atelectasis was observed in the CPAP group, although heterogeneity between studies was substantial (RR 0.62, 95% CI 0.45 to 0.86; I(2) = 61%). Pneumonia was reported in five studies, including 563 participants; CPAP reduced the rate of pneumonia, and no important heterogeneity was noted (RR 0.43, 95% CI 0.21 to 0.84; I(2) = 0%). The number of participants identified as having serious hypoxia was reported in two studies, with no clear difference between CPAP and control groups, given imprecise results and substantial heterogeneity between trials (RR 0.48, 95% CI 0.22 to 1.02; I(2) = 67%). A reduced rate of reintubation was reported in the CPAP group compared with the control group in two studies, and no important heterogeneity was identified (RR 0.14, 95% CI 0.03 to 0.58; I(2) = 0%). Admission into the intensive care unit (ICU) for invasive ventilation and supportive care was reduced in the CPAP group, but this finding did not reach statistical significance (RR 0.45, 95% CI 0.18 to 1.14; I(2) = 0).Secondary outcomes such as length of hospital stay and adverse effects were only minimally reported.A summary of findings table was constructed using the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) principle. The quality of evidence was determined to be very low. AUTHORS' CONCLUSIONS: Very low-quality evidence from this review suggests that CPAP initiated during the postoperative period might reduce postoperative atelectasis, pneumonia and reintubation, but its effects on mortality, hypoxia or invasive ventilation are uncertain. Evidence is not sufficiently strong to confirm the benefits or harms of CPAP during the postoperative period in those undergoing major abdominal surgery. Most of the included studies did not report on adverse effects attributed to CPAP.New, high-quality research is much needed to evaluate the use of CPAP in preventing mortality and morbidity following major abdominal surgery. With increasing availability of CPAP to our surgical patients and its potential to improve outcomes (possibly in conjunction with intraoperative lung protective ventilation strategies), unanswered questions regarding its efficacy and safety need to be addressed. Any future study must report on the adverse effects of CPAP.

The type 2 anti-Müllerian hormone receptor has splice variants that are dominant-negative inhibitors.

Anti-Müllerian hormone (AMH) has both paracrine and hormonal actions that occur at different AMH concentrations, and in cells with different densities of its specific receptor (Amhr2). This diversity is not explained by canonical AMH signaling. We report that Amhr2 has two splice variants: Amhr2Δ2 (AMH binding site) and Amhr2Δ9/10 (kinase domain). Both spliced variants inhibit AMH signaling in a reporter assay. The mRNA for the spliced variants was relatively less abundant than Amhr2 mRNA in all tissues. This suggests that the physiological function(s) of the receptor variants may be restricted to specific cellular/subcellular sites and/or to the transport of AMH.

A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons.

Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z'-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency.