protein complexes

Mass spectrometry-based shotgun proteomic analysis of C. elegan

Insights into the pre-initiation events of bacteriophage phi6 RNA-dependent RNA polymerase : towards the assembly of a productive binary complex

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.Peer reviewe

Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate

RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production

The consolidated European synthesis of CH4 and N2O emissions for the European Union and United Kingdom : 1990-2017

Reliable quantification of the sources and sinks of greenhouse gases, together with trends and uncertainties, is essential to monitoring the progress in mitigating anthropogenic emissions under the Paris Agreement. This study provides a consolidated synthesis of CH4 and N2O emissions with consistently derived state-of-the-art bottom-up (BU) and top-down (TD) data sources for the European Union and UK (EU27 C UK). We integrate recent emission inventory data, ecosystem process-based model results and inverse modeling estimates over the period 1990-2017. BU and TD products are compared with European national greenhouse gas inventories (NGHGIs) reported to the UN climate convention UNFCCC secretariat in 2019. For uncertainties, we used for NGHGIs the standard deviation obtained by varying parameters of inventory calculations, reported by the member states (MSs) following the recommendations of the IPCC Guidelines. For atmospheric inversion models (TD) or other inventory datasets (BU), we defined uncertainties from the spread between different model estimates or model-specific uncertainties when reported. In comparing NGHGIs with other approaches, a key source of bias is the activities included, e.g., anthropogenic versus anthropogenic plus natural fluxes. In inversions, the separation between anthropogenic and natural emissions is sensitive to the geospatial prior distribution of emissions. Over the 2011-2015 period, which is the common denominator of data availability between all sources, the anthropogenic BU approaches are directly comparable, reporting mean emissions of 20.8 TgCH(4) yr (-1) (EDGAR v5.0) and 19.0 TgCH(4) yr(-1) (GAINS), consistent with the NGHGI estimates of 18.9 +/- 1.7 TgCH(4) yr(-1). The estimates of TD total inversions give higher emission estimates, as they also include natural emissions. Over the same period regional TD inversions with higher-resolution atmospheric transport models give a mean emission of 28.8 TgCH(4) yr(-1). Coarser-resolution global TD inversions are consistent with regional TD inversions, for global inversions with GOSAT satellite data (23.3 TgCH(4) yr(-1)) and surface network (24.4 TgCH(4) yr (-1)). The magnitude of natural peatland emissions from the JSBACH-HIMMELI model, natural rivers and lakes emissions, and geological sources together account for the gap between NGHGIs and inversions and account for 5.2 TgCH(4) yr(-1). For N2O emissions, over the 2011-2015 period, both BU approaches (EDGAR v5.0 and GAINS) give a mean value of anthropogenic emissions of 0.8 and 0.9 TgN(2)Oyr(-1), respectively, agreeing with the NGHGI data (0.9 0.6 TgN(2)Oyr(-1)). Over the same period, the average of the three total TD global and regional inversions was 1.3 +/- 0.4 and 1.3 +/- 0.1 TgN(2)Oyr(-1), respectively. The TD and BU comparison method defined in this study can be operationalized for future yearly updates for the calculation of CH4 and N2O budgets both at the EU CUK scale and at the national scale.Peer reviewe

Will emergency and surgical patients participate in and complete alcohol interventions? A systematic review

<p>Abstract</p> <p>Background</p> <p>In the everyday surgical life, staff may experience that patients with Alcohol Use Disorders (AUDs) seem reluctant to participate in alcohol intervention programs. The objective was therefore to assess acceptance of screening and intervention as well as adherence to the intervention program among emergency department (ED) and surgical patients with AUDs.</p> <p>Methods</p> <p>A systematic literature search was followed by extraction of acceptance and adherence rates in ED and surgical patients. Numbers needed to screen (NNS) were calculated. Subgroup analyses were carried out based on different study characteristics.</p> <p>Results</p> <p>The literature search revealed 33 relevant studies. Of these, 31 were randomized trials, 28 were conducted in EDs and 31 evaluated the effect of brief alcohol intervention. Follow-up was mainly conducted after six and/or twelve months.</p> <p>Four in five ED patients accepted alcohol screening and two in three accepted participation in intervention. In surgical patients, two in three accepted screening and the intervention acceptance rate was almost 100%. The adherence rate was above 60% for up to twelve months in both ED and surgical patients. The NNS to identify one eligible AUD patient and to get one eligible patient to accept participation in alcohol intervention varied from a few up to 70 patients.</p> <p>The rates did not differ between randomized and non-randomized trials, brief and intensive interventions or validated and self-reported alcohol consumption. Adherence rates were not affected by patients' group allocation and type of follow-up.</p> <p>Conclusions</p> <p>Most emergency and surgical patients with AUD accept participation in alcohol screening and interventions and complete the intervention program.</p

The consolidated European synthesis of CH4 and N2O emissions for the European Union and United Kingdom : 1990-2019

Funding Information: We thank Aurélie Paquirissamy, Géraud Moulas and the ARTTIC team for the great managerial support offered during the project. FAOSTAT statistics are produced and disseminated with the support of its member countries to the FAO regular budget. Annual, gap-filled and harmonized NGHGI uncertainty estimates for the EU and its member states were provided by the EU GHG inventory team (European Environment Agency and its European Topic Centre on Climate change mitigation). Most top-down inverse simulations referred to in this paper rely for the derivation of optimized flux fields on observational data provided by surface stations that are part of networks like ICOS (datasets: 10.18160/P7E9-EKEA , Integrated Non-CO Observing System, 2018a, and 10.18160/B3Q6-JKA0 , Integrated Non-CO Observing System, 2018b), AGAGE, NOAA (Obspack Globalview CH: 10.25925/20221001 , Schuldt et al., 2017), CSIRO and/or WMO GAW. We thank all station PIs and their organizations for providing these valuable datasets. We acknowledge the work of other members of the EDGAR group (Edwin Schaaf, Jos Olivier) and the outstanding scientific contribution to the VERIFY project of Peter Bergamaschi. Timo Vesala thanks ICOS-Finland, University of Helsinki. The TM5-CAMS inversions are available from https://atmosphere.copernicus.eu (last access: June 2022); Arjo Segers acknowledges support from the Copernicus Atmosphere Monitoring Service, implemented by the European Centre for Medium-Range Weather Forecasts on behalf of the European Commission (grant no. CAMS2_55). This research has been supported by the European Commission, Horizon 2020 Framework Programme (VERIFY, grant no. 776810). Ronny Lauerwald received support from the CLand Convergence Institute. Prabir Patra received support from the Environment Research and Technology Development Fund (grant no. JPMEERF20182002) of the Environmental Restoration and Conservation Agency of Japan. Pierre Regnier received financial support from the H2020 project ESM2025 – Earth System Models for the Future (grant no. 101003536). David Basviken received support from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (METLAKE, grant no. 725546). Greet Janssens-Maenhout received support from the European Union's Horizon 2020 research and innovation program (CoCO, grant no. 958927). Tuula Aalto received support from the Finnish Academy (grants nos. 351311 and 345531). Sönke Zhaele received support from the ERC consolidator grant QUINCY (grant no. 647204).Peer reviewedPublisher PD

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Small non-coding RNAs mount a silent revolution in gene expression

During the last decade, it has become evident that small non-coding RNAs (ncRNAs) participate in widespread and essential regulatory mechanisms in most eukaryotic cells. Novel classes of small RNAs, their biogenesis pathways and cellular effects are continuously being described, and new properties of already established ncRNAs are still being discovered. As the list of small RNA molecules and their roles becomes more and more extensive, one can get lost in the midst of new information. In this review, we attempt to bring order to the small ncRNA transcriptome by covering some of the major milestones of recent years. We go through many of the new properties that have been attributed to already familiar RNA molecules, and introduce some of the more recent novel classes of tiny ncRNAs

In vitro activities of the multifunctional RNA silencing polymerase QDE-1 of Neurospora crassa

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases