The tubercular badger and the uncertain curve:- The need for a multiple stressor approach in environmental radiation protection.

This article presents the results of a workshop held in Stirling, Scotland in June 2018, called to examine critically the effects of low-dose ionising radiation on the ecosphere. The meeting brought together participants from the fields of low- and high-dose radiobiology and those working in radioecology to discuss the effects that low doses of radiation have on non-human biota. In particular, the shape of the low-dose response relationship and the extent to which the effects of low-dose and chronic exposure may be predicted from high dose rate exposures were discussed. It was concluded that high dose effects were not predictive of low dose effects. It followed that the tools presently available were deemed insufficient to reliably predict risk of low dose exposures in ecosystems. The workshop participants agreed on three major recommendations for a path forward. First, as treating radiation as a single or unique stressor was considered insufficient, the development of a multidisciplinary approach is suggested to address key concerns about multiple stressors in the ecosphere. Second, agreed definitions are needed to deal with the multiplicity of factors determining outcome to low dose exposures as a term can have different meanings in different disciplines. Third, appropriate tools need to be developed to deal with the different time, space and organisation level scales. These recommendations permit a more accurate picture of prospective risks.

Taking action on overuse: Creating the culture for change.

BACKGROUND: Unnecessary care contributes to high costs and places patients at risk of harm. While most providers support reducing low-value care, changing established practice patterns is difficult and requires active engagement in sustained behavioral, organizational, and cultural change. Here we describe an action-planning framework to engage providers in reducing overused services. METHODS: The framework is informed by a comprehensive review of social science theory and literature, published reports of successful and unsuccessful efforts to reduce low-value care, and interviews with innovators of value-based care initiatives in twenty-three health care organizations across the United States. A multi-stakeholder advisory committee provided feedback on the framework and guidance on optimizing it for use in practice. RESULTS: The framework describes four conditions necessary for change: prioritize addressing low-value care; build a culture of trust, innovation and improvement; establish shared language and purpose; and commit resources to measurements. These conditions foster productive sense-making conversations between providers, between providers and patients, and among members of the health care team about the potential for harm from overuse and reflection on current frequency of use. Through these conversations providers, patients and team members think together as a group, learn how to coordinate individual behaviors, and jointly develop possibilities for coordinated action around specific areas of overuse. CONCLUSIONS: Organizational efforts to engage providers in value-based care focused on creating conditions for productive sense-making conversations that lead to change. IMPLICATIONS: Organizations can use this framework to enhance and strengthen provider engagement efforts to do less of what potentially harms and more of what truly helps patients.

Structure of human dual-specificity phosphatase 7, a potential cancer drug target.

Human dual-specificity phosphatase 7 (DUSP7/Pyst2) is a 320-residue protein that belongs to the mitogen-activated protein kinase phosphatase (MKP) subfamily of dual-specificity phosphatases. Although its precise biological function is still not fully understood, previous reports have demonstrated that DUSP7 is overexpressed in myeloid leukemia and other malignancies. Therefore, there is interest in developing DUSP7 inhibitors as potential therapeutic agents, especially for cancer. Here, the purification, crystallization and structure determination of the catalytic domain of DUSP7 (Ser141-Ser289/C232S) at 1.67 Å resolution are reported. The structure described here provides a starting point for structure-assisted inhibitor-design efforts and adds to the growing knowledge base of three-dimensional structures of the dual-specificity phosphatase family.

Structural determinants of tobacco vein mottling virus protease substrate specificity.

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Šresolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

Aestuariibacter aggregatus sp. nov., a moderately halophilic bacterium isolated from seawater of the Yellow Sea.

A Gram-negative, non-spore-forming, catalase- and oxidase-positive, strictly aerobic, short rod-shaped bacterium with a single, polar flagellum, designated strain WH169(T), was isolated from seawater of the Yellow Sea in China. Buds and prosthecae were formed when the organism was grown at 20 degrees C for 12 days on marine 2216E agar. The organism grew optimally at 37 degrees C, in pH 7.0-8.0, and in the presence of 4.0-6.0% w/v NaCl. Growth did not occur in a medium without Na(+) or sea salts. Strain WH169(T) contained ubiquinone-8 as the predominant respiratory lipoquinone and C(16:1)omega7c and/or C(16:1)omega6c (35.9%), C(16:0) (25.3%) and C(18:1)omega7c (9.7%) as the major fatty acids. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. The DNA G+C content of strain WH169(T) was 49.4 mol%. 16S rRNA gene sequence analysis showed that strain WH169(T) showed 95.1% sequence similarity to both type strains of the only two species in the genus Aestuariibacter. On the basis of the polyphasic taxonomic evidence presented in this study, it was concluded that strain WH169(T) should be classified as a novel species of Aestuariibacter, for which the name Aestuariibacter aggregatus sp. nov. is proposed, with the type strain WH169(T) (=CGMCC 1.8995(T)=LMG 25283(T)).

Vibrio atypicus sp. nov., isolated from the digestive tract of the Chinese prawn (Penaeus chinensis O'sbeck).

A culture (designated strain HHS02(T)) was isolated from Chinese prawn (Penaeus chinensis, O'sbeck) and determined to be a member of the genus Vibrio. Strain HHS02(T) comprised slightly curved, rod-shaped, non-endospore-forming, Gram-negative, catalase-negative, oxidase-positive, O/129-sensitive and facultatively anaerobic cells that were motile by means of a single polar flagellum. Growth of strain HHS02(T) occurred in 0.5-7 % (w/v) NaCl [optimally in 1-3 % (w/v) NaCl] and between pH 7.0 and 10.0 (optimally at pH 8.0-9.0). The strain showed growth between 16 and 30 °C (optimum 20 °C). Analysis using the 16S rRNA, gapA, gyrB, mreB, pyrH, recA and topA gene sequences of the novel isolate revealed that the organism belonged to the genus Vibrio, with ∼98, 98, 90, 88, 92, 89 and 83 % sequence similarity, respectively, with representatives of the genus Vibrio. DNA-DNA hybridization experiments indicated that the novel strain was distinct from recognized species of the genus Vibrio. The major fatty acid components were summed feature 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH, 38.7 %), C(16 : 0) (22.9 %) and C(18 : 1)ω7c (12.5 %). The G+C content of the genomic DNA was 44.4 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, it is concluded that strain HHS02(T) should be classified as a novel species of the genus Vibrio, for which the name Vibrio atypicus sp. nov. is proposed. The type strain is HHS02(T) (=CGMCC 1.8461(T)=LMG 24781(T)).

Structural basis for binding of RNA and cofactor by a KsgA methyltransferase.

Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli.

Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.

Sneathiella chinensis gen. nov., sp. nov., a novel marine alphaproteobacterium isolated from coastal sediment in Qingdao, China.

The taxonomic position of strain LMG 23452(T), which was isolated from coastal sediment from an aquaculture site near Qingdao, China, in 2000, was determined. Strain LMG 23452(T) comprised Gram-negative, non-spore-forming, motile rods and was found to be a halotolerant, aerobic, chemoheterotroph that produces catalase and oxidase. Comparative 16S rRNA gene sequence analysis revealed that strain LMG 23452(T) shared approximately 89 % sequence similarity with members of the genera Devosia, Hyphomonas, Ensifer and Chelatococcus, which belong to two different orders within the Alphaproteobacteria. Further phylogenetic analysis of the 16S rRNA gene sequence showed that strain LMG 23452(T) formed a separate branch within the order Rhizobiales, falling between the genera Devosia and Ensifer of the families Hyphomicrobiaceae and Rhizobiaceae, respectively. Strain LMG 23452(T) could be differentiated from its closest phylogenetic neighbours on the basis of several phenotypic features, including hydrolysis of the substrates starch and casein and assimilation of the carbohydrates d-glucose, d-mannose, mannitol, maltose and l-arabinose, and chemotaxonomically by the presence of the fatty acids C(14 : 0) 3-OH, C(16 : 1)omega11c, C(16 : 1)omega5c and C(18 : 1)omega5c. The major fatty acids detected in strain LMG 23452(T) were C(18 : 1)omega7c, C(16 : 0), C(19 : 0) cyclo omega8c, C(16 : 1)omega7c and C(17 : 1)omega6c and the G+C content of the genomic DNA was 57.1 mol%. Therefore, the polyphasic data support the placement of strain LMG 23452(T) within a novel genus and species, for which the name Sneathiella chinensis gen. nov., sp. nov. is proposed. The type strain is LMG 23452(T) (=CBMAI 737(T)).

Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics.

Understanding how the various host cells respond to probiotic bacteria in vitro may provide important insight into elaborate immune responses triggered by beneficial bacteria. The aim of this study was to investigate the detailed pattern of the mRNA expression of cytokines (IL-1beta, IL-8, TNF-alpha and TGF-beta) in head kidney (HK) leucocytes and gut cells isolated from rainbow trout (Oncorhynchus mykiss Walbaum) after co-culturing with live probiotics. HK leucocytes and gut cells adjusted to 5 x 10(6) and 2 x 10(6) ml(-1), respectively, in L-15 medium containing 25% decomplemented FCS and 300 mg l(-1) L-glutamine were co-cultured with Carnobacterium maltaromaticum B26 and C. divergens B33 at an multiplicity of infection of 25 for 6 and 12 h. Quantitative real-time reverse transcriptase polymerase chain reaction using SYBR Green I was employed to determine the mRNA expression of studied genes. Although neither probiotic strains significantly induced mRNA of the cytokines in gut cells, expression ratios of IL-1beta and TNF-alpha of HK cells were significantly higher, suggesting that these bacteria can stimulate innate immunity in rainbow trout.

The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

A novel bacteriocin-like substance (BLIS) from a pathogenic strain of Vibrio harveyi.

Inter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaemolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, trypsin, pronase E, SDS and incubation at > or =60 degrees C for 10 min. The activity was stable between pH 2-11 for at least 5 h. Anion-exchange chromatography, gel filtration, SDS-PAGE and two-dimensional gel electrophoresis revealed the presence of a single major peak, comprising a protein with a pI of approximately 5.4 and a molecular mass of approximately 32 kDa. The N-terminal amino acid sequence of the protein comprised Asp-Glu-Tyr-Ile-Ser-X-Asn-Lys-X-Ser-Ser-Ala-Asp-Ile (with X representing cysteine or modified amino acid residues). A similarity search based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) generated peptide masses and the N-terminal sequence did not yield any significant matches.