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    Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass
    (Portland Press on behalf of the Biochemical Society, 2018-10) Sterritt OW; Lang EJM; Kessans SA; Ryan TM; Demeler B; Jameson GB; Parker EJ
    In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.
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    Retract p < 0.005 and propose using JASP, instead
    (F1000Research, 12/12/2017) Perezgonzalez JD; Frías-Navarro MD
    Seeking to address the lack of research reproducibility in science, including psychology and the life sciences, a pragmatic solution has been raised recently: to use a stricter p < 0.005 standard for statistical significance when claiming evidence of new discoveries. Notwithstanding its potential impact, the proposal has motivated a large mass of authors to dispute it from different philosophical and methodological angles. This article reflects on the original argument and the consequent counterarguments, and concludes with a simpler and better-suited alternative that the authors of the proposal knew about and, perhaps, should have made from their Jeffresian perspective: to use a Bayes factors analysis in parallel (e.g., via JASP) in order to learn more about frequentist error statistics and about Bayesian prior and posterior beliefs without having to mix inconsistent research philosophies.
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    Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls
    (Wiley, 3/09/2013) Lee KJD; Cornuault VRG; Manfield IW; Ralet MC; Knox JP
    Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG-I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG-I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG-I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.
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    The deconstruction of pectic rhamnogalacturonan I unmasks the occurrence of a novel arabinogalactan oligosaccharide epitope
    (Oxford University Press (OUP), 1/11/2015) Buffetto F; Cornuault VRG; Rydahl MG; Ropartz D; Alvarado C; Echasserieau V; Le Gall S; Bouchet B; Tranquet O; Verhertbruggen Y; Willats WGT; Knox JP; Ralet MC; Guillon F
    Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.
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    Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case
    (John Wiley & Sons, 2017) Brown S; Norris GE