Rocaglates as dual-targeting agents for experimental cerebral malaria

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.We thank Susan Gauthier, Genevieve Perreault, and Patrick Senechal for technical assistance. This work was supported by a research grant (to P.G.) from the Canadian Institutes of Health Research (CIHR) (Foundation Grant). J.P. and P.G. are supported by a James McGill Professorship salary award. D.L. is supported by fellowships from the Fonds de recherche sante Quebec, the CIHR Neuroinflammation training program. J.P. is supported by CIHR Research Grant FDN-148366. M.S. is supported by a CIHR Foundation grant. J.A.P. is supported by NIH Grant R35 GM118173. Work at the Boston University Center for Molecular Discovery is supported by Grant R24 GM111625. K.C.K. was supported by a CIHR Foundation Grant and the Canada Research Chair program. (Canadian Institutes of Health Research (CIHR); James McGill Professorship salary award; Fonds de recherche sante Quebec; CIHR Neuroinflammation training program; FDN-148366 - CIHR Research Grant; CIHR Foundation grant; R35 GM118173 - NIH; Canada Research Chair program; R24 GM111625

The isolation of novel "Erwinia" phages and their use in the study of bacterial phytopathogenicity

A number of bacteriophages were isolated on the "soft rot" phytopathogens Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Several of these phages were used to obtain phage resistant mutants of SCRI1043, in order to investigate the role of the bacterial cell surface in virulence. While a number of phenotypic properties relating to pathogenicity and virulence of this strain have already been uncovered, little is known about the role of the cell surface in virulence. It was hoped that the use of phages would allow selection of mutants altered in both cell surface and virulence. Two phage resistant mutants, A5/22 and A5/8, exhibited reduced virulence when inoculated into potato plants, and were investigated further. Both mutants showed pleiotropic phenotypes. As well as reduced virulence and phage resistance, these mutants showed a number of other phenotypic alterations including, a reduction in the production of plant cell wall degrading enzymes, increased sensitivity to surface active agents, alterations in lipopolysaccharide and outer membrane protein profiles and reduced motility. A5/22 also exhibited bacteriostasis in the presence of galactose. Mutant A5/22 was more severely affected in its virulence than A5/8, which reflected in its greater deviation from the wild type phenotype. While no one phenotypic alteration could be directly associated with the reduced virulence of either mutant, a combination of several phenotypes may have been responsible. The phages isolated in this study were the first reported for these strains of Erwinia, and were therefore characterised under a number of criteria. All phages were grouped on the basis of structural morphology, restriction endonuclease digestion and host range. This is the first detailed characterisation of phages for Erwinia carotovora subsp. atroseptica. All isolated phages were tested for generalised transduction, a method of molecular genetic analysis so far unavailable to Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Two phages, ØKP and ØMl, were capable of generalised transduction in SCRI193 and SCRI1043 respectively. Both these phages were characterised and transducing frequencies improved. ØMl is the first transducing phage reported for Erwinia carotovora subsp. atroseptica and ØKP is only the second for Erwinia carotovora subsp. carotovora. Both phages are now being used extensively in the laboratory

The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu

The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu

Structural Diversity in Bacterial Ribosomes: Mycobacterial 70S Ribosome Structure Reveals Novel Features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria

Active ribosome profiling with RiboLace

Summary: Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution. : Clamer et al. present RiboLace, a method for isolating active ribosomes and associated proteins, intact mRNAs, or ribosome-protected fragments. RiboLace accurately quantifies translation levels, providing positional data of active ribosomes with nucleotide resolution. Requiring lower input than current ribosome profiling protocols, RiboLace can be used with challenging biological samples. Keywords: ribosome profiling, translation, puromycin, translational control, protein synthesis, ribosome, proteome, polysomal profiling, translatom

Double J/ψJ/\psi production in pion-nucleon scattering at COMPASS

We present the study of the production of double $J/\psi$ mesons using COMPASS data collected with a 190 GeV/$c$ $\pi^-$ beam scattering off NH$_{3}$, Al and W targets. Kinematic distributions of the collected double $J/\psi$ events are analysed, and the double $J/\psi$ production cross section is estimated for each of the COMPASS targets. The results are compared to predictions from single- and double-parton scattering models as well as the pion intrinsic charm and the tetraquark exotic resonance hypotheses. It is demonstrated that the single parton scattering production mechanism gives the dominant contribution that is sufficient to describe the data. An upper limit on the double intrinsic charm content of pion is evaluated. No significant signatures that could be associated with exotic tetraquarks are found in the double $J/\psi$ mass spectrum.Comment: 12 pages, 4 figure

Light isovector resonances in π-p →π-π-π+p at 190 GeV/c

We have performed the most comprehensive resonance-model fit of π-π-π+ states using the results of our previously published partial-wave analysis (PWA) of a large data set of diffractive-dissociation events from the reaction π-+p→π-π-π++precoil with a 190 GeV/c pion beam. The PWA results, which were obtained in 100 bins of three-pion mass, 0.5<2.5 GeV/c2, and simultaneously in 11 bins of the reduced four-momentum transfer squared, 0.1<1.0 (GeV/c)2, are subjected to a resonance-model fit using Breit-Wigner amplitudes to simultaneously describe a subset of 14 selected waves using 11 isovector light-meson states with JPC=0-+, 1++, 2++, 2-+, 4++, and spin-exotic 1-+ quantum numbers. The model contains the well-known resonances π(1800), a1(1260), a2(1320), π2(1670), π2(1880), and a4(2040). In addition, it includes the disputed π1(1600), the excited states a1(1640), a2(1700), and π2(2005), as well as the resonancelike a1(1420). We measure the resonance parameters mass and width of these objects by combining the information from the PWA results obtained in the 11 t′ bins. We extract the relative branching fractions of the ρ(770)π and f2(1270)π decays of a2(1320) and a4(2040), where the former one is measured for the first time. In a novel approach, we extract the t′ dependence of the intensity of the resonances and of their phases. The t′ dependence of the intensities of most resonances differs distinctly from the t′ dependence of the nonresonant components. For the first time, we determine the t′ dependence of the phases of the production amplitudes and confirm that the production mechanism of the Pomeron exchange is common to all resonances. We have performed extensive systematic studies on the model dependence and correlations of the measured physical parameters

Searches for lepton-flavour-violating decays of the Higgs boson into eτ and μτ in \sqrt{s} = 13 TeV pp collisions with the ATLAS detector

Abstract This paper presents direct searches for lepton flavour violation in Higgs boson decays, H → eτ and H → μτ, performed using data collected with the ATLAS detector at the LHC. The searches are based on a data sample of proton-proton collisions at a centre-of-mass energy s $$\sqrt{s}$$ = 13 TeV, corresponding to an integrated luminosity of 138 fb−1. Leptonic (τ → ℓνℓντ) and hadronic (τ → hadrons ντ) decays of the τ-lepton are considered. Two background estimation techniques are employed: the MC-template method, based on data-corrected simulation samples, and the Symmetry method, based on exploiting the symmetry between electrons and muons in the Standard Model backgrounds. No significant excess of events is observed and the results are interpreted as upper limits on lepton-flavour-violating branching ratios of the Higgs boson. The observed (expected) upper limits set on the branching ratios at 95% confidence level, B $$\mathcal{B}$$ (H → eτ) < 0.20% (0.12%) and B $$\mathcal{B}$$ (H → μτ ) < 0.18% (0.09%), are obtained with the MC-template method from a simultaneous measurement of potential H → eτ and H → μτ signals. The best-fit branching ratio difference, B $$\mathcal{B}$$ (H → μτ) → B $$\mathcal{B}$$ (H → eτ), measured with the Symmetry method in the channel where the τ-lepton decays to leptons, is (0.25 ± 0.10)%, compatible with a value of zero within 2.5σ