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The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage β-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems. [ABSTRACT FROM AUTHOR]

The objective of the study described here was to determine basic plans and collaboration with first responder stakeholders and to identify perceived roles and responsibilities in preparing for and responding to a chemical disaster. A survey was developed and provided to environmental health personnel at county health departments (CHDs) in Florida. Most of the counties had good collaborative relationships with first responder stakeholders. A little more than half of the respondents had access to a resource manual with contact information and had developed and maintained a chemical plan. Rural counties were less likely to know “what to do” or their responsibility in a chemical disaster; however, both rural and nonrural counties were equally likely not to have a written plan. Public health agencies at the local CHD must be the communicators of public health messages in coordination with the incident commander and the state communications office in a chemical disaster, so it is important to strengthen collaboration and cooperation with chemical response stakeholders. [ABSTRACT FROM AUTHOR]

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-L-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 L-arabinofuranose (L-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-L-arabinofuranoside were also detected. After selective removal of α-1,3 L-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 L-Araf substituents, confirming the ability of SthAbf62A to remove α-L-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-D-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and L-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid L-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the L-arabinose binding pocket. [ABSTRACT FROM AUTHOR]

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Uslng a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. This central sheet is flanked by a set of three α-helices on one side and two helices on the other. The smaller domain is composed of an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, flanked by two oppositely oriented α-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase. [ABSTRACT FROM AUTHOR]

Background: We previously reported an association between 5HTTLPR genotype and outcome following cognitive-behavioural therapy (CBT) in child anxiety (Cohort 1). Children homozygous for the low-expression short-allele showed more positive outcomes. Other similar studies have produced mixed results, with most reporting no association between genotype and CBT outcome., Aims: To replicate the association between 5HTTLPR and CBT outcome in child anxiety from the Genes for Treatment study (GxT Cohort 2, n = 829)., Method: Logistic and linear mixed effects models were used to examine the relationship between 5HTTLPR and CBT outcomes. Mega-analyses using both cohorts were performed., Results: There was no significant effect of 5HTTLPR on CBT outcomes in Cohort 2. Mega-analyses identified a significant association between 5HTTLPR and remission from all anxiety disorders at follow-up (odds ratio 0.45, P = 0.014), but not primary anxiety disorder outcomes., Conclusions: The association between 5HTTLPR genotype and CBT outcome did not replicate. Short-allele homozygotes showed more positive treatment outcomes, but with small, non-significant effects. Future studies would benefit from utilising whole genome approaches and large, homogenous samples., (© The Royal College of Psychiatrists 2016.)