Characterisation of Pseudomonas rhamnolipids

The Gram negative organism, Pseudomonas aeruginosa, is often found in the lungs of patients with cystic fibrosis and other forms of severe bronchiectasis, where it secretes a number of extracellular toxins including the mono- and dirhamnolipids. The principal monorhamnolipid from P. aeruginosa has previously been identified as rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rh-C10.C10). A number of related mono- and dirhamnolipids have been purified from cultures of a clinical isolate of P. aeruginosa and identified by fast atom bombardment and electron impact mass spectrometry: these contain the 3-hydroxyoctanoyl-3-hydroxydecanoate (C8.C10) and 3-hydroxydecanoyl-3-hydroxydodecanoate (C10.C12) homologues. Structural isomers were also present where the order of the lipid linkage was transposed (Rh-C10.C8 and Rh-C12.C10). Unsaturated mono- and dirhamnolipids containing the 3-hydroxydecanoyl-3-hydroxydodec-5-enoate (C10.C12:1) lipid were also present.     

Gelled diet for screwworm (Diptera: Calliphoridae) mass production

A rearing system based on a diet gelled with Water-Lock G-400, a synthetic superabsorbent (poly(2-propenamide-co-2-propenoic acid, sodium salt)) (WL), was compared with the standard rearing system (liquid diet suspended in acetate fibers) for the mass production of screwworms, Cochliomyia hominivorax (Coquerel). The WL rearing system yielded 2% heavier pupae, 32% higher egg to pupa survival, and required 54% less diet and 88% less labor than the standard rearing system. Other advantages of the WL system include reduced susceptibility to suboptimal environmental conditions and labor practices and characteristics conducive to centralization and mechanization of rearing procedures.     

Comparative Phylogeography of Mississippi Embayment Fishes

The Mississippi Embayment is a prominent physiographic feature of eastern North America consisting of primarily lowland aquatic habitats and a fish fauna that is largely distinct from nearby highland regions. Numerous studies have demonstrated that both pre-Pleistocene and Pleistocene events have had a strong influence on the distributions and relationships of highland fishes in eastern North America. However, the extent to which these same events affected Embayment distributed taxa remains largely unexplored. The purpose of this study was to investigate the relative roles of pre-Pleistocene and Pleistocene events in shaping phylogeographic relationships of four stream dwelling fishes in the Mississippi Embayment. Molecular genetic analyses of the mitochondrial gene cytochrome b were performed for three ictalurid catfish species (Noturus miurus, n = 67; Noturus hildebrandi, n = 93, and Noturus phaeus, n = 44) and one minnow species (Cyprinella camura, n = 78), all distributed in tributary streams of the Mississippi Embayment. Phylogenetic relationships and divergence times among haplotypes for each species were estimated using maximum likelihood and Bayesian methods. Phylogenetic analyses recovered 6 major haplotype clades within N. miurus, 5 within N. hildbrandi, 8 within N. phaeus, and 8 within C. camura. All three Noturus species show a high degree of isolation by drainage, which is less evident in C. camura. A clade of haplotypes from tributaries in the southern portion of the Mississippi Embayment was consistently recovered in all four species. Divergence times among clades spanned the Pleistocene, Pliocene, and Miocene. Novel relationships presented here for C. camura and N. phaeus suggest the potential for cryptic species. Pre-Pleistocene and Pleistocene era sea level fluctuations coincide with some divergence events, but no single event explains any common divergence across all taxa. Like their highland relatives, a combination of both pre-Pleistocene and Pleistocene era events have driven divergences among Embayment lineages.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

A new mechanism for the aerobic catabolism of dimethyl sulfide.

Aerobic degradation of dimethyl sulfide (DMS), previously described for thiobacilli and hyphomicrobia, involves catabolism to sulfide via methanethiol (CH3SH). Methyl groups are sequentially eliminated as HCHO by incorporation of O2 catalyzed by DMS monooxygenase and methanethiol oxidase. H2O2 formed during CH3SH oxidation is destroyed by catalase. We recently isolated Thiobacillus strain ASN-1, which grows either aerobically or anaerobically with denitrification on DMS. Comparative experiments with Thiobacillus thioparus T5, which grows only aerobically on DMS, indicate a novel mechanism for aerobic DMS catabolism by Thiobacillus strain ASN-1. Evidence that both organisms initially attacked the methyl group, rather than the sulfur atom, in DMS was their conversion of ethyl methyl sulfide to ethanethiol. HCHO transiently accumulated during the aerobic use of DMS by T. thioparus but not with Thiobacillus strain ASN-1. Catalase levels in cells grown aerobically on DMS were about 100-fold lower in Thiobacillus strain ASN-1 than in T. thioparus T5, suggesting the absence of H2O2 formation during DMS catabolism. Also, aerobic growth of T. thioparus T5 on DMS was blocked by the catalase inhibitor 3-amino-1,2,4-triazole whereas that of Thiobacillus strain ASN-1 was not. Methyl butyl ether, but not CHCl3, blocked DMS catabolism by T. thioparus T5, presumably by inhibiting DMS monooxygenase and perhaps methanethiol oxidase. In contrast, DMS metabolism by Thiobacillus strain ASN-1 was unaffected by methyl butyl ether but inhibited by CHCl3. DMS catabolism by Thiobacillus strain ASN-1 probably involves methyl transfer to a cobalamin carrier and subsequent oxidation as folate-bound intermediates.     

The total burden of rare,non-synonymous exome genetic variants is not associated with childhood or late-life cognitive ability

Human cognitive ability shows consistent, positive associations with fitness components across the life-course. Underlying genetic variation should therefore be depleted by selection, which is not observed. Genetic variation in general cognitive ability (intelligence) could be maintained by a mutation–selection balance, with rare variants contributing to its genetic architecture. This study examines the association between the total number of rare stop-gain/loss, splice and missense exonic variants and cognitive ability in childhood and old age in the same individuals. Exome array data were obtained in the Lothian Birth Cohorts of 1921 and 1936 (combined N = 1596). General cognitive ability was assessed at age 11 years and in late life (79 and 70 years, respectively) and was modelled against the total number of stop-gain/loss, splice, and missense exonic variants, with minor allele frequency less than or equal to 0.01, using linear regression adjusted for age and sex. In both cohorts and in both the childhood and late-life models, there were no significant associations between rare variant burden in the exome and cognitive ability that survived correction for multiple testing. Contrary to our a priori hypothesis, we observed no evidence for an association between the total number of rare exonic variants and either childhood cognitive ability or late-life cognitive ability.