Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

Engineering poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer composition in E. coli

A strain of Escherichia coli was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) of specified composition between 5% and 18% HV. A gene encoding propionyl-CoA synthetase (prpE from S. enterica) was placed under the control of the IPTG-inducible tac promoter (P(taclacUV5)) while the polyhydroxyalkanoate synthesis operon (phaBCA) from R. eutropha was expressed constitutively. A strain of E. coli harboring both plasmids was grown in defined medium and PHBV was produced with specified hydroxyvalerate (HV) molar content between 5% and 18%. The molecular weight of the copolymer was approximately 700,000 across various HV contents, and average polydispersity was approximately 1.3. The majority of the PHBV production occurred during the late exponential/stationary phase. The HV content of the copolymer generally peaked early in the incubation before falling to its final value. We found that the time profiles of PrpE activity, propionyl-CoA, and acetyl-CoA were well correlated to the HV content time profile. Despite an abundance of propionyl-CoA, incorporation of HV into the copolymer was inefficient. Therefore, both the PHA operon and conditions affecting the availability of propionyl-CoA must be chosen carefully to achieve the desired HV content. The ability to engineer copolymer composition control into an E. coli strain would be useful in cases where the feedstock composition is not adjustable.     

THE IMPORTANCE OF MOSQUITO BEHAVIOURAL ADAPTATIONS TO MALARIA CONTROL IN AFRICA

Over the past decade the use of long‐lasting insecticidal nets (LLINs), in combination with improved drug therapies, indoor residual spraying (IRS), and better health infrastructure, has helped reduce malaria in many African countries for the first time in a generation. However, insecticide resistance in the vector is an evolving threat to these gains. We review emerging and historical data on behavioral resistance in response to LLINs and IRS. Overall the current literature suggests behavioral and species changes may be emerging, but the data are sparse and, at times unconvincing. However, preliminary modeling has demonstrated that behavioral resistance could have significant impacts on the effectiveness of malaria control. We propose seven recommendations to improve understanding of resistance in malaria vectors. Determining the public health impact of physiological and behavioral insecticide resistance is an urgent priority if we are to maintain the significant gains made in reducing malaria morbidity and mortality.     

Fabrication of Bioinspired Structured Superhydrophobic and Superoleophilic Copper Mesh for Efficient Oil-water Separation

Oily water treatment has attracted the attention of many researchers.The development of effective and cheap oil/water separation materials is urgent for treating this problem.Herein,inspired by superhydrophobic typical plant leaves such as lotus,red rose and marigold,superhydrophobic and superoleophilic copper mesh was fabricated by etching and then surface modification with 1-dodecanethiol (HS(CH2)11CH3).A rough silver layer is formed on the mesh surface after immersion.The obtained mesh surface exhibits superhydrophobicity and superoleophilicity and the static water contact angle was 153° ± 3°.In addition,the as-prepared copper mesh shows self-cleaning character with water and chemical stability.The as-prepared copper foam can easily remove the organic solvents either on water or underwater.We demonstrate that by using the as-prepared mesh,oils can be absorbed and separated,and that high separation efficiencies of larger than 92％ are retained for various oils.Thus,such superhydrophobic and superoleophilic copper mesh is a very promising material for the application of oil spill cleanup and industrial oily wastewater treatment.     

Can we measure ecological sustainability? Landscape pattern as an indicator for naturalness and land use intensity at regional,national and European level

European landscapes have been shaped over the centuries by processes related to human land use, which are reflected in regionally distinct landscape patterns. Since landscape pattern has been linked to biodiversity and other ecological values of the landscapes, this paper explores landscape pattern as a tool for ecological sustainability assessments at the regional (Austrian Cultural Landscapes), national (Austria) and European (European Union + Norway, Switzerland) level with focus on agricultural landscapes. A set of landscape metrics served as a basis to assess naturalness and geometrisation of Austrian and European landscapes as a proxy for their sustainability. To achieve an accurate spatially explicit assessment, we applied a spatial reference framework consisting in units that are homogeneous in biophysical and socio-economic contexts, adapted the regional approach for its application at European level, and developed relative sustainability thresholds for the landscape metrics. The analyses revealed that several landscape metrics, particularly the “Number of Shape Characterising Points” showed a high correlation with the degree of naturalness. The sustainability map of Austria based on an ordinal regression model revealed well-known problem regions of ecological sustainability. At the European level, the relative deviation from the average pattern showed clearly the simplification processes in the landscapes. However, a better spatial resolution of land cover data would add to the refinement of pattern analysis in regions and therefore the assessment of sustainability. We recommend the combination of information of different scales for the formulation and implementation of sustainability policies.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5

The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin. Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis. Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases. This led us to suspect the presence of a contaminant in the purified samples. Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography. Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity. These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.