@article{8262, abstract = {Background: The genus Burkholderia consists of species that occupy remarkably diverse ecological niches. Its best known members are important pathogens, B. mallei and B. pseudomallei, which cause glanders and melioidosis, respectively. Burkholderia genomes are unusual due to their multichromosomal organization, generally comprised of 2-3 chromosomes. Results: We performed integrated genomic analysis of 127 Burkholderia strains. The pan-genome is open with the saturation to be reached between 86,000 and 88,000 genes. The reconstructed rearrangements indicate a strong avoidance of intra-replichore inversions that is likely caused by selection against the transfer of large groups of genes between the leading and the lagging strands. Translocated genes also tend to retain their position in the leading or the lagging strand, and this selection is stronger for large syntenies. Integrated reconstruction of chromosome rearrangements in the context of strains phylogeny reveals parallel rearrangements that may indicate inversion-based phase variation and integration of new genomic islands. In particular, we detected parallel inversions in the second chromosomes of B. pseudomallei with breakpoints formed by genes encoding membrane components of multidrug resistance complex, that may be linked to a phase variation mechanism. Two genomic islands, spreading horizontally between chromosomes, were detected in the B. cepacia group. Conclusions: This study demonstrates the power of integrated analysis of pan-genomes, chromosome rearrangements, and selection regimes. Non-random inversion patterns indicate selective pressure, inversions are particularly frequent in a recent pathogen B. mallei, and, together with periods of positive selection at other branches, may indicate adaptation to new niches. One such adaptation could be a possible phase variation mechanism in B. pseudomallei.}, author = {Bochkareva, Olga and Moroz, Elena V. and Davydov, Iakov I. and Gelfand, Mikhail S.}, issn = {1471-2164}, journal = {BMC Genomics}, publisher = {Springer Nature}, title = {{Genome rearrangements and selection in multi-chromosome bacteria Burkholderia spp.}}, doi = {10.1186/s12864-018-5245-1}, volume = {19}, year = {2018}, } @article{8265, abstract = {Genome rearrangements have played an important role in the evolution of Yersinia pestis from its progenitor Yersinia pseudotuberculosis. Traditional phylogenetic trees for Y. pestis based on sequence comparison have short internal branches and low bootstrap supports as only a small number of nucleotide substitutions have occurred. On the other hand, even a small number of genome rearrangements may resolve topological ambiguities in a phylogenetic tree. We reconstructed phylogenetic trees based on genome rearrangements using several popular approaches such as Maximum likelihood for Gene Order and the Bayesian model of genome rearrangements by inversions. We also reconciled phylogenetic trees for each of the three CRISPR loci to obtain an integrated scenario of the CRISPR cassette evolution. Analysis of contradictions between the obtained evolutionary trees yielded numerous parallel inversions and gain/loss events. Our data indicate that an integrated analysis of sequence-based and inversion-based trees enhances the resolution of phylogenetic reconstruction. In contrast, reconstructions of strain relationships based on solely CRISPR loci may not be reliable, as the history is obscured by large deletions, obliterating the order of spacer gains. Similarly, numerous parallel gene losses preclude reconstruction of phylogeny based on gene content.}, author = {Bochkareva, Olga and Dranenko, Natalia O. and Ocheredko, Elena S. and Kanevsky, German M. and Lozinsky, Yaroslav N. and Khalaycheva, Vera A. and Artamonova, Irena I. and Gelfand, Mikhail S.}, issn = {2167-8359}, journal = {PeerJ}, publisher = {PeerJ}, title = {{Genome rearrangements and phylogeny reconstruction in Yersinia pestis}}, doi = {10.7717/peerj.4545}, volume = {6}, year = {2018}, } @article{8274, abstract = {Background/Aim: Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT). Materials and Methods: Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry. Results: Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68+ macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells. Conclusion: Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT.}, author = {Carvalho, Maria Isabel and Bianchini, Rodolfo and Fazekas-Singer, Judit and Herrmann, Ina and Flickinger, Irene and Thalhammer, Johann G. and Pires, Isabel and Jensen-Jarolim, Erika and Queiroga, Felisbina L.}, issn = {1791-7530}, journal = {Anticancer Research}, number = {5}, pages = {2811--2817}, publisher = {International Institute of Anticancer Research}, title = {{Bidirectional regulation of COX-2 expression between cancer cells and macrophages}}, doi = {10.21873/anticanres.12525}, volume = {38}, year = {2018}, } @inproceedings{8298, abstract = {Sharding, or partitioning the system’s state so that different subsets of participants handle it, is a proven approach to building distributed systems whose total capacity scales horizontally with the number of participants. Many distributed ledgers have adopted this approach to increase their performance, however, they focus on the permissionless setting that assumes the existence of a strong adversary. In this paper, we deploy channels for permissioned blockchains. Our first contribution is to adapt sharding on asset-management applications for the permissioned setting, while preserving liveness and safety even on transactions spanning across-channels. Our second contribution is to leverage channels as a confidentiality boundary, enabling different organizations and consortia to preserve their privacy within their channels and still be part of a bigger collaborative ecosystem. To make our system concrete we map it on top of Hyperledger Fabric.}, author = {Androulaki, Elli and Cachin, Christian and De Caro, Angelo and Kokoris Kogias, Eleftherios}, booktitle = {Computer Security}, isbn = {9783319990729}, issn = {0302-9743}, location = {Barcelona, Spain}, pages = {111--131}, publisher = {Springer Nature}, title = {{Channels: Horizontal scaling and confidentiality on permissioned blockchains}}, doi = {10.1007/978-3-319-99073-6_6}, volume = {11098}, year = {2018}, } @inproceedings{8297, abstract = {Designing a secure permissionless distributed ledger (blockchain) that performs on par with centralized payment processors, such as Visa, is a challenging task. Most existing distributed ledgers are unable to scale-out, i.e., to grow their totalprocessing capacity with the number of validators; and those that do, compromise security or decentralization. We present OmniLedger, a novel scale-out distributed ledger that preserves longterm security under permissionless operation. It ensures security and correctness by using a bias-resistant public-randomness protocol for choosing large, statistically representative shards that process transactions, and by introducing an efficient crossshard commit protocol that atomically handles transactions affecting multiple shards. OmniLedger also optimizes performance via parallel intra-shard transaction processing, ledger pruning via collectively-signed state blocks, and low-latency “trust-butverify” validation for low-value transactions. An evaluation ofour experimental prototype shows that OmniLedger’s throughput scales linearly in the number of active validators, supporting Visa-level workloads and beyond, while confirming typical transactions in under two seconds.}, author = {Kokoris Kogias, Eleftherios and Jovanovic, Philipp and Gasser, Linus and Gailly, Nicolas and Syta, Ewa and Ford, Bryan}, booktitle = {2018 IEEE Symposium on Security and Privacy}, isbn = {9781538643532}, issn = {2375-1207}, location = {San Francisco, CA, United States}, pages = {583--598}, publisher = {IEEE}, title = {{OmniLedger: A secure, scale-out, decentralized ledger via sharding}}, doi = {10.1109/sp.2018.000-5}, year = {2018}, } @article{8443, abstract = {Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent–membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states.}, author = {Kurauskas, Vilius and Hessel, Audrey and Ma, Peixiang and Lunetti, Paola and Weinhäupl, Katharina and Imbert, Lionel and Brutscher, Bernhard and King, Martin S. and Sounier, Rémy and Dolce, Vincenza and Kunji, Edmund R. S. and Capobianco, Loredana and Chipot, Christophe and Dehez, François and Bersch, Beate and Schanda, Paul}, issn = {1948-7185}, journal = {The Journal of Physical Chemistry Letters}, keywords = {General Materials Science}, number = {5}, pages = {933--938}, publisher = {American Chemical Society}, title = {{How detergent impacts membrane proteins: Atomic-level views of mitochondrial carriers in dodecylphosphocholine}}, doi = {10.1021/acs.jpclett.8b00269}, volume = {9}, year = {2018}, } @article{8440, abstract = {Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death.}, author = {Weinhäupl, Katharina and Brennich, Martha and Kazmaier, Uli and Lelievre, Joel and Ballell, Lluis and Goldberg, Alfred and Schanda, Paul and Fraga, Hugo}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Biochemistry, Molecular Biology}, number = {22}, pages = {8379--8393}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis}}, doi = {10.1074/jbc.ra118.002251}, volume = {293}, year = {2018}, } @article{8442, abstract = {Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.}, author = {Chipot, Christophe and Dehez, François and Schnell, Jason R. and Zitzmann, Nicole and Pebay-Peyroula, Eva and Catoire, Laurent J. and Miroux, Bruno and Kunji, Edmund R. S. and Veglia, Gianluigi and Cross, Timothy A. and Schanda, Paul}, issn = {0009-2665}, journal = {Chemical Reviews}, keywords = {General Chemistry}, number = {7}, pages = {3559--3607}, publisher = {American Chemical Society}, title = {{Perturbations of native membrane protein structure in alkyl phosphocholine detergents: A critical assessment of NMR and biophysical studies}}, doi = {10.1021/acs.chemrev.7b00570}, volume = {118}, year = {2018}, } @article{8441, abstract = {Solid-state near-rotary-resonance measurements of the spin–lattice relaxation rate in the rotating frame (R1ρ) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of 15N R1ρ data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, “dead time” in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays. The multiple 15N R1ρ measurements at different SL fields and temperatures have been conducted in 1D mode (i.e. without site-specific resolution) for a set of four different microcrystalline protein samples (GB1, SH3, MPD-ubiquitin and cubic-PEG-ubiquitin) to study the overall protein rocking in a crystal. While the amplitude of this motion varies very significantly, its correlation time for all four sample is practically the same, 30–50 μs. The amplitude of the rocking motion correlates with the packing density of a protein crystal. It has been suggested that the rocking motion is not diffusive but likely a jump-like dynamic process.}, author = {Krushelnitsky, Alexey and Gauto, Diego and Rodriguez Camargo, Diana C. and Schanda, Paul and Saalwächter, Kay}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, number = {1}, pages = {53--67}, publisher = {Springer Nature}, title = {{Microsecond motions probed by near-rotary-resonance R1ρ 15N MAS NMR experiments: The model case of protein overall-rocking in crystals}}, doi = {10.1007/s10858-018-0191-4}, volume = {71}, year = {2018}, } @article{8439, abstract = {Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of Gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E. coli and pathogenic P. aeruginosa with long O-antigen polysaccharides assembled in solution as vesicles or elongated micelles. Solid-state NMR with magic-angle spinning permitted the identification of NMR signals arising from regions with different flexibilities in the LPS, from the lipid components to the O-antigen polysaccharides. Atomic scale data on the LPS enabled the study of the interaction of gentamicin antibiotic bound to P. aeruginosa LPS, for which we could confirm that a specific oligosaccharide is involved in the antibiotic binding. The possibility to study LPS alone and bound to a ligand when it is assembled in membrane-like structures opens great prospects for the investigation of proteins and antibiotics that specifically target such an important molecule at the surface of Gram-negative bacteria.}, author = {Laguri, Cedric and Silipo, Alba and Martorana, Alessandra M. and Schanda, Paul and Marchetti, Roberta and Polissi, Alessandra and Molinaro, Antonio and Simorre, Jean-Pierre}, issn = {1554-8929}, journal = {ACS Chemical Biology}, keywords = {Molecular Medicine, Biochemistry, General Medicine}, number = {8}, pages = {2106--2113}, publisher = {American Chemical Society}, title = {{Solid state NMR studies of intact lipopolysaccharide endotoxin}}, doi = {10.1021/acschembio.8b00271}, volume = {13}, year = {2018}, }