The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana

INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast-localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2’s role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C-to-U RNA editing, altered accumulation of chloroplast transcripts and chloroplast-encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next-generation sequencing (RIP-seq), and the diversity of RNA species identified supports ISE2’s involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non-canonical Ski2-like RNA helicase that represents a separate sub-clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2’s evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression

Vaccinia virus immunomodulator A46 : a lipid and protein-binding scaffold for sequestering host TIR-domain proteins

TS received Austrian Science Fund (FWF) grants P24038, W1221 and W1258. GAB is a member of Max F. Perutz Laboratories and the Vienna International PostDoctoral Program (VIPS). TKS is a holder of Wellcome Trust grant 097831. IU has Spanish Ministry of Economy and Competitiveness grant BIO2013-49604-EXP.Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N-and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.Publisher PDFPeer reviewe

Comparative Genome Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112T and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112T has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B12). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with 13C3-glycerol demonstrated that L. reuteri JCM 1112T could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112T to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri

Hematopoietic Cell–Restricted Deletion of CD36 Reduces High-Fat Diet–Induced Macrophage Infiltration and Improves Insulin Signaling in Adipose Tissue

OBJECTIVE: The fatty acid translocase and scavenger receptor CD36 is important in the recognition and uptake of lipids. Accordingly, we hypothesized that it plays a role in saturated fatty acid-induced macrophage lipid accumulation and proinflammatory activation. RESEARCH DESIGN AND METHODS: In vitro, the effect of CD36 inhibition and deletion in lipid-induced macrophage inflammation was assessed using the putative CD36 inhibitor, sulfosuccinimidyl oleate (SSO), and bone marrow-derived macrophages from mice with (CD36KO) or without (wild-type) global deletion of CD36. To investigate whether deletion of macrophage CD36 would improve insulin sensitivity in vivo, wild-type mice were transplanted with bone marrow from CD36KO or wild-type mice and then fed a standard or high-fat diet (HFD) for 20 weeks. RESULTS: SSO treatment markedly reduced saturated fatty acid-induced lipid accumulation and inflammation in RAW264.7 macrophages. Mice harboring CD36-specific deletion in hematopoietic-derived cells (HSC CD36KO) fed an HFD displayed improved insulin signaling and reduced macrophage infiltration in adipose tissue compared with wild-type mice, but this did not translate into protection against HFD-induced whole-body insulin resistance. Contrary to our hypothesis and our results using SSO in RAW264.7 macrophages, neither saturated fatty acid-induced lipid accumulation nor inflammation was reduced when comparing CD36KO with wild-type bone marrow-derived macrophages. CONCLUSIONS: Although CD36 does not appear important in saturated fatty acid-induced macrophage lipid accumulation, our study uncovers a novel role for CD36 in the migration of proinflammatory phagocytes to adipose tissue in obesity, with a concomitant improvement in insulin action

Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment

An evaluation of the exposure in nadir observation of the JEM-EUSO mission

We evaluate the exposure during nadir observations with JEM-EUSO, the Extreme Universe Space Observatory,on-board the Japanese Experiment Module of the International Space Station. Designed as a mission to explore the extreme energy Universe from space, JEM-EUSO will monitor the Earth's nighttime atmosphere to record the ultraviolet light from tracks generated by extensive air showers initiated by ultra-high energy cosmic rays. In the present work, we discuss the particularities of space-based observation and we compute the annual exposure in nadir observation. The results are based on studies of the expected trigger aperture and observational duty cycle, as well as, on the investigations of the effects of clouds and different types of background light. We show that the annual exposure is about one order of magnitude higher than those of the presently operating ground-based observatories.Fil: Adams, J. H.. University of Alabama in Huntsville; Estados UnidosFil: Ahmad, S.. Universite Paris Sud; FranciaFil: Albert, J. N..Fil: Allard, D.. Universite Paris Diderot - Paris 7; FranciaFil: Ambrosio, M.. Istituto Nazionale di Fisica Nucleare; ItaliaFil: Anchordoqui, L.. Medical College Of Wisconsin; Estados UnidosFil: Anzalone, A.. INAF; ItaliaFil: Arai, Y.. High Energy Accelerator Research Organization (KEK); JapónFil: Aramo, C..Fil: Asano, K.. Interactive Research Center of Science, Tokyo Institute of Technology; JapónFil: Ave, M.. Universidad de Santiago de Compostela; EspañaFil: Barrillon, P.. Universite de Paris; FranciaFil: Batsch, T.. National Centre for Nuclear Research; PoloniaFil: Bayer, J.. University of Tubingen; AlemaniaFil: Belenguer, T.. j Instituto Nacional de Técnica Aeroespacial (INTA); EspañaFil: Bellotti, R.. Universita’ degli Studi di Bari Aldo Moro and INFN; ItaliaFil: Berlind, A. A.. Vanderbilt University; Estados UnidosFil: Bertaina, M.. Universita di Torino; ItaliaFil: Biermann, P. L.. Karlsruhe Institute of Technology (KIT); AlemaniaFil: Biktemerova,. Joint Institute for Nuclear Research; RusiaFil: Blaksley, C.. Universite de la Sorbona Nouvelle; FranciaFil: Blecki, J.. Space Research Centre of the Polish Academy of Sciences (CBK); PoloniaFil: Blin-Bondil, S.. Universite de Paris; FranciaFil: Blumer, J.. Karlsruhe Institute of Technology (KIT),; AlemaniaFil: Bobik, P.. Institute of Experimental Physics; EslovaquiaFil: Bogomilov, M.. St. Kliment Ohridski University of Sofia; BulgariaFil: Bonamente, M.. University of Alabama in Huntsville; Estados UnidosFil: Briz, S.. Universidad Carlos III de Madrid,; EspañaFil: Supanitsky, Alberto Daniel. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; Argentin

Performances of JEM-EUSO

In this paper we describe the requirements and the expected performances of JEM-EUSO. Designed as the first mission to explore the Ultra High Energy Universe from space, JEM-EUSO will monitor the earth's atmosphere at night to record the UV (300–400 nm) tracks generated by the Extensive Air Showers produced by Ultra High Energy primaries propagating in the atmosphere. After briefing summarizing the main aspects of the JEM-EUSO Instrument and mission baseline, we will present, in details, our studies of the expected trigger rate, the estimated exposure, as well as on the expected angular, energy, and Xmax resolution. Eventually, the obtained results will be discussed in the context of the scientific requirements of the mission

Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein—Barr Virus Latent Phase Protein LMP1

© 2017, Springer Science+Business Media, LLC. Intracellular fragments of latent phase protein LMP1 of Epstein—Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein

Science of atmospheric phenomena with JEM-EUSO

The main goal of the JEM-EUSO experiment is the study of Ultra HighEnergy Cosmic Rays (UHECR, 10^19 - 10^21 eV ), but the method which will be used (detection of the secondary light emissions induced by cosmic rays in the atmosphere) allows to study other luminous phenomena. The UHECRs will be detected through the measurement of the emission in the range between 290 and 430 m, where some part of Transient Luminous Events (TLEs) emission also appears. This work discusses the possibility of using the JEM-EUSO Telescope to get new scientific results on TLEs. The high time resolution of this instrument allows to observe the evolution of TLEs with great precision just at the moment of their origin. Thepaper consists of four parts: review of the present knowledge on the TLE, presentation of the results of the simulations of the TLE images in the JEM-EUSO telescope, results of the Russian experiment Tatiana-2 and discussion of the possible progress achievable in this field with JEM-EUSO as well as possible cooperation with other space projects devoted to the study of TLE-TARANIS and ASIM. In atmospheric physics, the study of TLEs became one of the main physical subjects of interest after their discovery in 1989. In the years 1992 - 1994 detection was performed fromsatellite, aircraft and space shuttle and recently from the International Space Station. These events have short duration (milliseconds) and small scales (km to tens of km) and appear at altitudes 50 - 100 km. Their nature is still not clear and each new experimental data can be useful for a better understanding of these mysterious phenomena.Fil: Adams, J. H.. University of Alabama in Huntsville; Estados UnidosFil: Ahmad, S.. Ecole Polytechnique; FranciaFil: Albert, J. N.. Univ Paris-Sud; FranciaFil: Allard, D.. Univ Paris Diderot; FranciaFil: Anchordoqui, L.. University of Wisconsin-Milwaukee; Estados UnidosFil: Andreev, V.. University of California; Estados UnidosFil: Anzalone, A.. INAF - Istituto di Astrofisica Spaziale e Fisica Cosmica di Palermo; ItaliaFil: Arai, Y.. High Energy Accelerator Research Organization (KEK); JapónFil: Asano, K.. Tokyo Institute of Technology; JapónFil: Ave Pernas, M.. Universidad de Alcala (UAH); EspañaFil: Barrillon, P.. Univ Paris-Sud; FranciaFil: Batsch, T.. Skobeltsyn Institute of Nuclear Physics; RusiaFil: Bayer, J.. University of Tubingen; AlemaniaFil: Bechini, R.. Universita’ di Torino; ItaliaFil: Belenguer, T.. Instituto Nacional de Tecnica Aeroespacial (INTA); EspañaFil: Bellotti, R.. Istituto Nazionale di Fisica Nucleare; ItaliaFil: Belov, K.. University of California; Estados UnidosFil: Berlind, A. A.. Vanderbilt University; Estados UnidosFil: Bertaina, M.. Istituto Nazionale di Fisica Nucleare; ItaliaFil: Biermann, P. L.. Karlsruhe Institute of Technology (KIT); AlemaniaFil: Biktemerova, S.. Joint Institute for Nuclear Research; RusiaFil: Blaksley, C.. Univ Paris Diderot; FranciaFil: Blanc, N.. Swiss Center for Electronics and Microtechnology (CSEM); SuizaFil: Blecki, J.. Space Research Centre of the Polish Academy of Sciences (CBK; PoloniaFil: Blin-Bondil, S.. Ecole Polytechnique; FranciaFil: Blumer, J.. Karlsruhe Institute of Technology (KIT),; AlemaniaFil: Bobik, P.. Institute of Experimental Physics; EslovaquiaFil: Bogomilov, M.. University of Sofia; BulgariaFil: Bonamente, M.. University of Alabama in Huntsville; Estados UnidosFil: Supanitsky, Alberto Daniel. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: The JEM-EUSO Collaboration

QM/MM description of newly selected catalytic bioscavengers against organophosphorus compounds revealed reactivation stimulus mediated by histidine residue in the acyl-binding loop

© 2018 Zlobin, Mokrushina, Terekhov, Zalevsky, Bobik, Stepanova, Aliseychik, Kartseva, Panteleev, Golovin, Belogurov, Gabibov and Smirnov. Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pKa, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme's active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons