Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.     

Topographic and biotic regulation of aboveground carbon storage in subtropical broad-leaved forests of Taiwan

There is a growing need to understand, and ultimately manage, carbon storage by forest ecosystems. Broad-leaved evergreen forests of Taiwan provide an outstanding opportunity to examine factors that regulate ecosystem carbon storage. We utilized data from three Taiwan Forest Dynamics Plots (FS, LHC, and PTY) in which every tree is identified, measured, tagged and mapped, to examine factors regulating carbon storage as estimated from aboveground biomass. Allometric equations were used to estimate the aboveground biomass of each tree, and a model building procedure was used to examine relationships between plot-level aboveground biomass (AGB; Mg/ha) and a suite of topographic and biotic factors. We found that our study sites have AGB values comparable to some of the most carbon dense forests in the world. Across all three sites, maximum biomass was contained in the taxonomic families Fagaceae, Lauraceae and Theaceae. In the FS site, we identified slope convexity (P = 0.03) and elevation (P < 0.001) as topographic predictors of AGB and found that maximum AGB was found in topographically flat areas. In FS, stem density (P < 0.001) was a significant biotic predictor of AGB and the maxima occurred at intermediate densities. In LHC, we found that convexity (P < 0.001) and slope (P < 0.001) were significantly related to AGB which was maximized along a topographic ridge in the plot. Species richness (P < 0.001) was a significant biotic predictor of AGB in LHC, and the relationship indicated slightly higher AGB at higher levels of species richness. The only significant factor related to AGB in PTY was species richness (P = 0.02). Further work is needed to seek a mechanistic understanding of topographic factors and species richness as drivers of carbon storage in forests.     

A genomic view of the human-Bacteroides thetaiotaomicron symbiosis

The human gut is colonized with a vast community of indigenous microorganisms that help shape our biology. Here, we present the complete genome sequence of the Gram-negative anaerobe Bacteroides thetaiotaomicron, a dominant member of our normal distal intestinal microbiota. Its 4779-member proteome includes an elaborate apparatus for acquiring and hydrolyzing otherwise indigestible dietary polysaccharides and an associated environment-sensing system consisting of a large repertoire of extracytoplasmic function sigma factors and one- and two-component signal transduction systems. These and other expanded paralogous groups shed light on the molecular mechanisms underlying symbiotic host-bacterial relationships in our intestine.     

Gamma-ray flares from the Crab Nebula

A young and energetic pulsar powers the well-known Crab Nebula. Here, we describe two separate gamma-ray (photon energy greater than 100 mega-electron volts) flares from this source detected by the Large Area Telescope on board the Fermi Gamma-ray Space Telescope. The first flare occurred in February 2009 and lasted approximately 16 days. The second flare was detected in September 2010 and lasted approximately 4 days. During these outbursts, the gamma-ray flux from the nebula increased by factors of four and six, respectively. The brevity of the flares implies that the gamma rays were emitted via synchrotron radiation from peta-electron-volt (10(15) electron volts) electrons in a region smaller than 1.4 × 10(-2) parsecs. These are the highest-energy particles that can be associated with a discrete astronomical source, and they pose challenges to particle acceleration theory.     

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.     

Relationships between Cd concentrations in different vegetables and those in arable soils,and food safety evaluation of vegetables in Taiwan

Abstract

The cadmium (Cd) accumulation capacity of various vegetables and the relationship between Cd concentrations in edible parts of vegetables and those in soils were assessed by conducting field experiments at Cd-contaminated sites in northern and central Taiwan. In addition, to thoroughly assess Cd concentrations in vegetables and to understand the food safety of vegetables in Taiwan, 2257 paired vegetable and surface soil samples were collected from major vegetable production areas for Cd concentration analysis. According to the bioconcentration factors calculated, the Cd accumulation capacity varied considerably among the vegetable species tested, and the order of the five vegetables with the highest capacities is peanut (Arachis hypogaea L.) > amaranth (Amaranthus tricolor L.) > spinach (Spinacia oleraceae L.) > gynura (Gynura bicolor DC.) > okra (Hibiscus esculentus L.), whereas the order of the five vegetables with the lowest capacities is bitter gourd (Momordica charantia L.) < cucumber (Cucumis sativus L.) < asparagus bean (Vigna unguiculata (L.) Walp. ssp. sesquipedalis (L.) Verdc.) < snap bean (Phaseolus vulgaris L.) < sponge gourd (Luffa cylindrica Roem.). We derived 29 soil–plant transfer models of Cd for individual vegetable species based on available pools of the Cd, manganese (Mn), zinc (Zn), copper (Cu) and iron (Fe) concentrations; soil pH; and cation exchange capacity (CEC). According to the derived models, the available Cd, Mn and Zn concentrations, and pH, served as the main factors affecting Cd concentrations in the edible parts of vegetables, whereas the CEC and available Cu and Fe concentrations are less important factors. The data of previous studies and those of this study from major vegetable production areas, including 30 vegetable crops, were used to evaluate the safety of vegetables in Taiwan. The results indicated that the percentage of vegetables with Cd concentrations exceeding the regulatory concentration was 0.54%; therefore, the food safety concern is low. However, 9.8, 1.0, 0.9 and 0.6% of amaranth, cabbage (Brassica oleracea L. Capitata Group), Chinese cabbage (Brassica pekinensis Skeels.) and carrot (Daucus carota L.), respectively, had Cd concentrations in the edible parts exceeding the regulatory concentration. Particular attention should be paid when planning production areas for these vegetables. We recommend cultivating peanuts in fields with a soil Cd concentration < 0.33 mg kg^?1. The bioconcentration factors and soil–plant transfer models derived in this study might serve as assessment tools for planning farming areas for these vegetables.