Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose

Transient receptor potential cation channel, subfamily V, member 4 and airway sensory afferent activation: Role of adenosine triphosphate

BackgroundSensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored.ObjectiveWe hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung.MethodsWe used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues.ResultsHere we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough.ConclusionThis study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough

The Impact of Recent European Droughts and Heatwaves on Trace Gas Surface Fluxes: Insights from Land Surface Data Assimilation

Heatwave and drought extremes can have significant impacts on vegetation, which can in turn lead to important effects on reactive trace gas fluxes at the land-atmosphere interface that can ultimately alter atmospheric composition. We present results from the EU-funded Sentinel EObased Emission and Deposition Service (SEEDS) project, which aimed at developing upgrades to the existing Copernicus Atmospheric Monitoring Service (CAMS) component on European air quality. In this work, we used land surface modelling (SURFEX – Surface Externalisée) combined with data assimilation (Extended Kalman Filter - EKF) of satellite leaf area index (LAI) to deliver improved estimation of the land surface state. The land surface model is coupled with an online model for dry deposition and an offline model (MEGANv3.1) for biogenic volatile organic compounds (BVOCs) to estimate trace gas losses and emissions, respectively. This approach exploits methods at the forefront of land surface modelling (dynamic vegetation simulation and data assimilation) and combines them with the latest algorithms to estimate trace gas fluxes at the surface. We present findings from two extreme events in Europe: the 2018 drought and the 2019 June/July heat waves. SURFEX was forced using ECMWF meteorology at 0.1° × 0.1° resolution that captured both events. Both extreme events provoked strong responses in the models for dry deposition velocity and BVOC emissions. The 2018 drought began in spring and endured through summer, during which dry deposition velocities declined steadily beyond seasonal norms due to increased stomatal resistance forced by the vegetation response to drought. Over continental Europe, BVOCs initially increased in the early phase of the drought, but then sharply declined into July in the worst-affected regions in Germany, Denmark, and Poland. Meanwhile, BVOCs increased in Scandinavia relative to seasonal norms due to the warmer-than-average conditions. The first episode of severe heat in 2019 arrived in late June, which initially caused a large increase in BVOC emissions compared to seasonal norms. Then drought set in during July and despite a second large heat wave BVOC emissions were lower overall compared to seasonal norms. In fact, the European-wide BVOC emissions were higher in June compared to July due to the drought effects that commenced later in the heat wave cycle. This reverses the normal seasonal cycle in BVOC emissions, and drought impacts on vegetation were the primary driver behind this. Dry deposition velocities are reduced during both heat waves, but we see a larger decline in the second heat wave in July when drought conditions are more severe. Our findings suggest that these impacts on trace gas surface fluxes would have a strong effect on atmospheric composition, and on photochemical ozone formation. We, therefore, conclude that these effects likely played a contributory role to the ozone pollution episodes that occurred coincidentally in time with the heat wave events in both 2018 and 2019. The project aim within SEEDS is to eventually test the BVOC emissions and dry deposition velocities within a chemical transport model participating within the CAMS regional ensemble (MOCAGE) and to therefore evaluate the impact on ozone

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

GEIA - The Global Emissions InitiAtive

two new programs are presently being linked within GEIA: ECCAD and CIERA. Their common aim is to facilitate access to emissions information. ECCAD (Emissions of chemical Compounds & Compilation of Ancillary Data, http://ether.ipsl. jussieu.fr/eccad) GEIA's new interactive emissions data portal that provides consistent access to global and regional emissions inventories and ancillary data, along with easy-to-use tools for analysis and visualization. CIERA (Community Initiative for Emissions Research & Applications, http://ciera-air.org/) is a GEIA community effort to develop interoperability in emissions datasets and tools, support evaluations of emissions inventories, and connect the emissions development and user communities. More details about ECCAD and CIERA are presented in the articles immediately following this note

Addressing Science and Policy Needs with Community Emissions Efforts

International audienceWe present community-driven emissions efforts within the Global Emissions InitiAtive (GEIA, http://www.geiacenter.org/), a joint IGAC/iLEAPS/AIMES initiative of the International Geosphere-Biosphere Programme. Since 1990, GEIA has served as a forum for the exchange of expertise and information on emissions. GEIA's mission is to (1) quantify anthropogenic emissions and natural exchanges of trace gases and aerosols; and (2) facilitate the use of this information by the research, assessment, and policy communities. GEIA supports a worldwide network of over 1200 developers and users in international scientific projects, providing a solid scientific foundation for atmospheric chemistry research. Moving forward, GEIA is broadening its role to serve the scientific, regulatory, and operational emission communities. GEIA intends to demonstrate the potential for improving emission information by promoting the interoperability of datasets and tools and by making use of near-real-time observations. As a first step toward these goals, two new programs are being linked with GEIA: * ECCAD (Emissions of Chemical Compounds & Compilation of Ancillary Data, http://eccad.sedoo.fr/) is GEIA's new interactive emissions data portal, providing consistent access to emission inventories and ancillary data with easy-to-use tools for analysis and visualization. * CIERA (Community Initiative for Emissions Research & Applications, http://ciera-air.org/) is a new GEIA community project to develop interoperability in emissions datasets and tools, support evaluations of inventories, communicate emissions information in innovative ways, and connect the emissions development and user communities. We invite the scientific and policy community to join the GEIA network and build partnerships to improve emissions information

Twenty-five years of continuous sulphur dioxide emission reduction in Europe

During the last twenty-five years European emission data have been compiled and reported under the Cooperative Programme for Monitoring and Evaluation of the Long-range Transmission of Air Pollutants in Europe (EMEP) as part of the work under the Convention on Long-range Transboundary Air Pollution (LRTAP). This paper presents emission trends of SO2 reported to EMEP and validated within the programme for the period 1980-2004. European sulphur emissions have been steadily decreasing over the last twenty-five years, amounting from about 55 Tg SO2 in 1980 to 15 Tg SO2 in 2004. The relative contribution of the European emissions to total global sulphur emissions has been halved during this period. Based on annual emission reports from European countries, three emission reduction regimes have been identified. The period 1980-1989 is characterized by low annual emission reductions (below 5% reduction per year and 20% for the whole period) and is dominated by emission reductions in Western Europe. The period 1990-1999 is characterised by high annual emission reductions (up to 11% reduction per year and 54% for the whole period), most pronounced in Eastern Europe. The annual emission reductions in the period 2000-2004 are medium to low and reflect the unified Europe, with equally large reductions in both East and West. The sulphur emission reduction has been largest in the Combustion in energy and transformation industries sector, but substantial decreases are also seen in the Non-industrial combustion plants together with Industrial Combustion and – Industrial Production Processes sectors. The majority of European countries have reduced their emissions by more than 60% between 1990 and 2004, and one quarter have already achieved sulphur emission reductions higher than 80%. At European level, the total sulphur target for 2010 set in the Gothenburg Protocol (16 Tg) has apparently already been met by 2004. However, still half of the Parties to the Gothenburg Protocol have to reduce further their sulphur emissions in order to attain their individual country total emission targets for 2010. It is also noteworthy that, contrasting the Gothenburg Protocol requirements, an increasing number of countries have recently been reporting increased sulphur emissions, while others report only minor decreases. The uncertainty in sulphur emission estimates is low, and although they are at the same level as recent reductions, the emission trends presented here are supported by different studies of air concentrations and depositions carried out within and outside the framework of the LTRAP Convention

Caliop near-real-time backscatter products compared to earlinet data

The expedited near-real-time Level 1.5 Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) version 3 products were evaluated against data from the ground-based European Aerosol Research Lidar Network (EARLINET). The statistical framework and results of the three-year evaluation of 48 CALIOP overpasses with ground tracks within a 100 km distance from operating EARLINET stations are presented and include analysis for the following CALIOP classifications of aerosol type: dust, polluted dust, clean marine, clean continental, polluted continental, mixed and/or smoke/biomass burning. For the complete data set comprising both the planetary boundary layer (PBL) and the free troposphere (FT) data, the correlation coefficient (R) was 0.86. When the analysis was conducted separately for the PBL and FT, the correlation coefficients were R = 0.6 and R = 0.85, respectively. From analysis of selected specific cases, it was initially thought that the presence of FT layers, with high attenuated backscatter, led to poor agreement of the PBL backscatter profiles between the CALIOP and EARLINET and prompted a further analysis to filter out such cases; however, removal of these layers did not improve the agreement as R reduced marginally from R = 0.86 to R = 0.84 for the combined PBL and FT analysis, increased marginally from R = 0.6 up to R = 0.65 for the PBL on its own, and decreased marginally from R = 0.85 to R = 0.79 for the FT analysis on its own. This suggests considerable variability, across the data set, in the spatial distribution of the aerosol over spatial scales of 100 km or less around some EARLINET stations rather than influence from elevated FT layers. For specific aerosol types, the correlation coefficient between CALIOP backscatter profiles and the EARLINET data ranged from R = 0.37 for polluted continental aerosol in the PBL to R = 0.57 for dust in the FT

The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.Gema Tarrasón, Mariona Aulí, Sanam Mustafa, Vladislav Dolgachev, Maria Teresa Domènech, Neus Prats, María Domínguez, Rosa López, Nuria Aguilar, Marta Calbet, Mercè Pont, Graeme Milligan, Steven L. Kunkel, Nuria Godessar