Impact of Extraction Methods on the Detectable Protein Complement of Metaproteomic Analyses of Marine Sediments

Metaproteomic analysis targets proteins, the catalytic entities in the habitat, thereby providing direct insights into the metabolic activity of the community studied. A major challenge still remaining for metaproteomics is the effective and comprehensive extraction of proteins from environmental samples, due to their high complexity with respect to organismic diversity and abundance range. Moreover, in certain habitats, the inherent matrix may interfere with protein extraction. In recent years, several studies reported different protein extraction methods for soils known for their complex geochemistry, but only three analyzed marine sediments that generally comprise different though similarly complex geochemistry. In this study, the impact of four different extraction methods was investigated for coastal North Sea and deep sea Pacific Ocean sediments. The extraction methods comprised (i) phenol, (ii) SDS, (iii) a mixture of SDS and phenol, and (iv) urea and thiourea. Prior to extraction, a cell and protein standard (CPS) was added to the sediment samples to trace recovery of proteins from different subcellular locations as well as dissolved BSA. While each extraction method detected distinct peptide complements, SDS‐phenol extraction generally achieved highest protein yield and most comprehensive CPS protein identification. Application of two different methods was shown to further improve proteome coverage.     

A different kind of day hospital--for patients with preterminal cancer and chronic disease.

A new day hospital has been set up in Sheffield for patients with preterminal cancer and chronic disease. During the first 26 months 197 patients with cancer and 66 chronically sick patients attended. Of the 2701 attendances by patients with cancer, breast cancer accounted for 38%. One hundred and forty of the patients with cancer died, 83% in the terminal-care unit and 12% at home. Ninety per cent of respondents to a questionnaire thought that the support provided was of great importance to both patient and family; and over two-thirds of the patients were said to have benefited from improved control of symptoms. A similar facility could be developed within existing day hospitals without major revenue consequences, and should produce a genuine improvement in care. This study suggests, however, that it would not shorten the terminal stay in hospital.     

Stereospecificity of amino acid hydroxamate inhibition of aminopeptidases

Hydroxamates of amino acids and aliphatic acids are effective inhibitors of Aeromonas proteolytica amino-peptidase (EC 3.4.11.10) and of both the cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases of swine kidney. Cytosolic leucine aminopeptidase and the Aeromonas enzyme were inhibited to a greater extent by D isomers than by the L enantiomorphs, manganese-activated kidney cytosolic leucine aminopeptidase being inhibited 10 times more effectively by D-leucine and D-valine hydroxamic acids than by the L isomers. The D isomers of these two compounds inhibited Aeromonas aminopeptidase to an even greater extent with Ki values of 2 X 10(-9) and 5 X 10(-9), respectively, whereas the corresponding L isomers were bound 150 times less tightly. With the Aeromonas enzyme, a comparison of inhibition by racemic mixtures with that of the corresponding L isomers indicated that in all cases the contribution of the D isomer was predominant. Isocaproic hydroxamic acid inhibited this enzyme equally well as L-leucine hydroxamic acid, indicating that the amino group orientation in the D isomer contributes to the binding efficacy. Swine kidney microsomal aminopeptidase was also inhibited by D isomers of leucine and valine hydroxamic acids but in contrast to the other two enzymes, the inhibition was 10-fold less than that observed for the corresponding L isomers. Cytosolic leucine aminopeptidase with either 6 g atoms of zinc per mol or 12 g atoms of zinc per mol was inhibited only slightly by any of the hydroxamic acid compounds; evidently enzyme-bound manganese (or magnesium) is specific for hydroxamate binding to this aminopeptidase.     

Fish passage design for sustainable hydropower in the temperate Southern Hemisphere: an evidence review

The development of hydropower and other infrastructure that disrupts river connectivity poses a serious threat to highly endemic and genetically distinct freshwater fish species in temperate parts of the Southern Hemisphere. Such locations have been neglected in previous reviews on fish passage. Fishways have long been constructed to mitigate the impacts of riverine barriers on fish, yet they have often failed for all but the largest, strongest swimming taxa. This is a particular problem in the temperate south, which is home to native species that are non-recreational and generally small-bodied with weak swimming abilities (e.g. Galaxiidae) relative to typical target species for fishway design (e.g. Salmonidae). Using the Eco Evidence method for rapid evidence synthesis, we undertook an assessment of evidence for effective fishway design focusing on species representative of the temperate south, including eel and lamprey. Systematic literature searches resulted in 630 publications. Through a rigorous screening process these were reduced to 46 publications containing 76 evidence items across 19 hypotheses relating to design criteria for upstream and downstream passage. We found an overwhelming lack of evidence for effective fishway design in the temperate south. Particular deficiencies were found with regard to the design of effective facilities for downstream passage. The attraction and entrance of upstream migrating fish into fishways is also relatively under-researched. Given the urgent need for effective fishways in the temperate south, these results justify an approach to fishway design based on a combination of empirical data and expert knowledge. In the meantime, significant resources should be assigned to improve the evidence base through high quality research. The particular deficiencies identified here could guide that research agenda.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.