Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function

GEO-6 assessment for the pan-European region

Through this assessment, the authors and the United Nations Environment Programme (UNEP) secretariat are providing an objective evaluation and analysis of the pan-European environment designed to support environmental decision-making at multiple scales. In this assessment, the judgement of experts is applied to existing knowledge to provide scientifically credible answers to policy-relevant questions. These questions include, but are not limited to the following:• What is happening to the environment in the pan-European region and why?• What are the consequences for the environment and the human population in the pan-European region?• What is being done and how effective is it?• What are the prospects for the environment in the future?• What actions could be taken to achieve a more sustainable future?<br/

First results from recent JET experiments in Hydrogen and Hydrogen-Deuterium plasmas

The hydrogen campaign completed at JET in 2016 has demonstrated isotope ratio control in JET-ILW using gas puffing and pellets for fuelling, Neutral Beam Injection alone or in combination, with D/H spectroscopy as a diagnostic. The plasma properties such as confinement, L-H threshold, density limit depend on the isotope composition. The L-H transition power increases with the hydrogen concentration with a wide plateau in the range 0.2<nH/(nD+nH)<0.8. Energy confinement is significantly lower in hydrogen than in comparable deuterium ELMy H-mode plasmas, suggesting an isotope mass scaling that is stronger than in IPB98(y,2). In L-mode, the isotope dependence of confinement is weaker. The H-mode density limit in hydrogen is up to 35% lower than in heuterium, whilst it is found to be higher in L-mode. The lower ion mass leads to reduced tungsten sputtering in hydrogen plasmas. During the campaign, the nD/(nD+nH) ratio dropped to ~1% in only a few discharges after the last deliberate introduction of deuterium, although it was seen to rise again to ~2% with several seconds of exposure of the divertor tiles to ~10MW of auxiliary heating. Several ICRH scenarios were also tested in hydrogen plasmas

A Velocity Based Boundary Element Method with Modified Trailing Edge for Prediction of the Partial Cavities on the Wings and Propeller Blades

This paper presents a new velocity based boundary element method for calculation of the partial cavities on the wings and propeller blades. The fully correct mathematical formulation of the problem about cavitating bodies is characteristic feature of the developed method and numerical algorithm. The special algorithm of Modified Trailing Edge (MTE) is used for definition of the lifting (circulation) part of the flow. MTE tool allows the approximate accounting for viscous effects on lift and pressure distribution. The miscellaneous cavity closure model is recommended for calculation. The prediction of the cavity patterns is performed using Iterative Cavity Alignment (ICA) procedure with Free Cavity Length (FCL). The results of the test calculations and comparisons with experimental data are given in the paper

Field-Dependent Effect of Crown Ether (18-Crown-6) on Ionic Conductance of α-Hemolysin Channels

Closing linear poly(ethylene glycol) (PEG) into a circular “crown” dramatically changes its dynamics in the α-hemolysin channel. In the electrically neutral crown ether (C(2)H(4)O)(6), six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend on voltage strongly and nonmonotonically. Within the whole frequency range accessible in channel reconstitution experiments, the noise power spectrum is “white”, showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel reaches its maximum (a few microseconds) at about the same voltage (∼100 mV, negative from the side of protein addition) where the crown's reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore

TLR2 and endosomal TLR-mediated secretion of IL-10 and immune suppression in response to phagosome-confined Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs

Progress on the PITZ TDS

A transverse deflecting system (TDS) is under commissioning at the Photo Injector Test Facility at DESY, Zeuthen site (PITZ). The structure was designed and manufactured by the Institute for Nuclear Research (INR RAS, Moscow, Russia) as prototype for the TDS in the injector part of the European XFEL. Last year the deflection voltage was limited for safety reasons, but after thorough investigations of the waveguide system we are now able to operate the cavity close to design specifications. The PITZ TDS streaks the electron beam vertically, allowing measurements of the longitudinal bunch profile, and, in combination with a subsequent horizontal bending magnet, also of the longitudinal phase space and slice energy spread. Furthermore, several quadrupole magnets and screen stations can be employed for slice emittance measurements using the TDS. This paper describes the progress in commissioning of the hardware, measurement techniques and simulations, and outlines the prospects of reliable slice emittance measurements at 20 MeV/c, where space charge forces complicate the determination of transfer matrices