Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.     

Proteome analysis of cultivated vascular smooth muscle cells from a CADASIL patient

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementing disease caused by mutations in the NOTCH3 gene, most which are missense mutations leading to an uneven number of cysteine residues in epidermal growth factor-like repeats in the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on the VSMCs of small and middle-sized arteries. Recent studies have demonstrated that impairment of Notch3 signaling is not the primary cause of the disease. In the present study we used proteomic analysis to characterize the protein expression pattern of a unique material of genetically genuine cultured human CADASIL VSMCs. We identified 11 differentially expressed proteins, which are involved in protein degradation and folding, contraction of VSMCs, and cellular stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic reticulum stress and activation of unfolded protein response, leading to increased reactive oxygen species and inhibition of cell proliferation. In addition, upregulation of contractile proteins suggests an alteration in the signaling system of VSMC contraction. The accumulation of N3ECD on the cell surface possibly upregulates the angiotensin II regulatory feedback loop and thereby enhances the readiness of the cells to respond to angiotensin II stimulation.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Characterization of a highly pathogenic<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain isolated from common cockchafer,<Emphasis Type="Italic">Melolontha melolontha</Emphasis>

A bacterial isolate (Mm2) of Melolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified as Bacillus thuringiensis subsp. tenebrionis. This isolate was compared to the reference strains by electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content and insecticidal activity. Cells of the isolate harbored flat square inclusions containing a protein component of approximately equal to65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of approximately equal to 50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate has cry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae of M. melolontha. The isolate Mm2 may be valuable as biological control agent for M. melolontha and other coleopteran insects.     

Cloning and Characterization of a Weissella confusa Dextransucrase and Its Application in High Fibre Baking

Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using ¹⁴C-sucrose radioisotope and reducing value-based assays, the former yielding K _m and V _max values of 14.7 mM and 8.2 μmol/(mg∙min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.     

The Glasgow consensus on the delineation between pesticide emission inventory and impact assessment for LCA

Purpose

Pesticides are applied to agricultural fields to optimise crop yield and their global use is substantial. Their consideration in life cycle assessment (LCA) is affected by important inconsistencies between the emission inventory and impact assessment phases of LCA. A clear definition of the delineation between the product system model (life cycle inventory—LCI, technosphere) and the natural environment (life cycle impact assessment—LCIA, ecosphere) is missing and could be established via consensus building.

Methods

A workshop held in 2013 in Glasgow, UK, had the goal of establishing consensus and creating clear guidelines in the following topics: (1) boundary between emission inventory and impact characterisation model, (2) spatial dimensions and the time periods assumed for the application of substances to open agricultural fields or in greenhouses and (3) emissions to the natural environment and their potential impacts. More than 30 specialists in agrifood LCI, LCIA, risk assessment and ecotoxicology, representing industry, government and academia from 15 countries and four continents, met to discuss and reach consensus. The resulting guidelines target LCA practitioners, data (base) and characterisation method developers, and decision makers.

Results and discussion

The focus was on defining a clear interface between LCI and LCIA, capable of supporting any goal and scope requirements while avoiding double counting or exclusion of important emission flows/impacts. Consensus was reached accordingly on distinct sets of recommendations for LCI and LCIA, respectively, recommending, for example, that buffer zones should be considered as part of the crop production system and the change in yield be considered. While the spatial dimensions of the field were not fixed, the temporal boundary between dynamic LCI fate modelling and steady-state LCIA fate modelling needs to be defined.

Conclusions and recommendations

For pesticide application, the inventory should report pesticide identification, crop, mass applied per active ingredient, application method or formulation type, presence of buffer zones, location/country, application time before harvest and crop growth stage during application, adherence with Good Agricultural Practice, and whether the field is considered part of the technosphere or the ecosphere. Additionally, emission fractions to environmental media on-field and off-field should be reported. For LCIA, the directly concerned impact categories and a list of relevant fate and exposure processes were identified. Next steps were identified: (1) establishing default emission fractions to environmental media for integration into LCI databases and (2) interaction among impact model developers to extend current methods with new elements/processes mentioned in the recommendations.

     

Seeing the trees for the leaves – oaks as mosaics for a host-specific moth

From the perspective of a specialist herbivore, how homogenous are individual tree crowns as patches of habitat? We partitioned variation in physical and chemical host leaf traits and in the abundance and performance of a specialist oak leaf miner, Tischeria ekebladella, into variation at different hierarchical levels. For the phenolic contents of the leaves, we examined variation among oak stands, among trees within stands and among branches within trees. For leaf size and water content, we assessed variation among trees within a single stand, among shoots within trees, and among leaves within shoots. For moth abundance and performance, we examined variation across all levels: among oak stands, among trees within stands, among branches within trees, among shoots within branches and among leaves and insect individuals. For measures of phenolic contents, we found little variation among stands but substantial variation among individual trees. Yet, a tree particularly rich in a given compound was often comparatively poor in another. At a finer spatial scale, the phenolic composition of individual parts of a single tree was quite consistent, whereas leaf weight and water content varied widely within individual tree crowns. Moth abundances varied more among shoots within branches than at any other spatial level, whereas moth survival showed equal levels of variation within individual shoots as among separate oak stands. Likewise, for four other measures of larval performance (assessed at the level of trees and lower), we found more variation within than between trees. In conclusion, the large variation observed in the performance of a specialist moth and in the physical traits of the leaves among different parts of single tree crowns refutes the image of an oak tree as an ‘island’ of internally homogeneous quality. Hence, we may expect little evolutionary adaptation of T. ekebladella at the scale of individual trees. The moths may instead evolve to behaviourally select their resource at a very fine scale. Large variation within trees also calls for extensive replication within trees in ecological sampling designs and/or the sampling of maximally similar leaves.