Insulation and wiring specificity of BceR‐like response regulators and their target promoters in Bacillus subtilis

BceRS and PsdRS are paralogous two‐component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P_bceA or P_psdA, resulting in a strong up‐regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross‐regulation has been observed between them. We therefore investigated the specificity determinants of P_bceA and P_psdA that ensure the insulation of these two paralogous pathways at the RR–promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high‐affinity, low‐specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low‐affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities.     

How do Mediterranean shrub species cope with shade? Ecophysiological response to different light intensities

• Under natural conditions, light exposure for Mediterranean shrubs can be highly variable, especially during cloudy days or under a canopy, and can interfere with other environmental factors such as temperature and water availability.
• With the aim of decoupling the effect of radiation and temperature from water availability, we conducted an experiment where two perennial and three summer semi‐deciduous shrub species were subjected to different levels of irradiation. In order to follow plant responses to light exposure, we measured gas exchange, photosystem II photochemical efficiency, photosynthetic pigments and leaf mass area in spring and summer.
• Results showed that all study species presented a plastic response to different light conditions, and that light‐related traits varied in a coordinated manner. Summer semi‐deciduous species exhibited a more opportunistic response, with higher photosynthesis rates in full sun, but under shade conditions, the two strategies presented similar assimilation rates. Stomatal conductance did not show such a drastic response as photosynthetsis, being related to changes in WUE. Daily cycles of F_v/F_m revealed a slight photoinhibitory response during summer, mainly in perennial species. In all cases photosynthetic pigments adjusted to the radiation level; leaves had lower chlorophyll content, higher pool of xanthophylls and higher proportion of the de‐epoxydaded state of xanthophylls under sun conditions. Lutein content increased in relation to the xanthophyll pool under shade conditions.
• Our results evidenced that radiation is an important driving factor controlling morphological and physiological status of Mediterranean shrub species, independently of water availability. Summer semi‐deciduous species exhibit a set of traits with higher response variability, maximising their photosynthetic assimilation under different sun conditions.

     

Landscape simplification increases vineyard pest outbreaks and insecticide use

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13‐year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four‐fold in simplified, vineyard‐dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi‐natural habitats. Similarly, insecticide applications doubled in vineyard‐dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest‐control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.     

Liquid–liquid extraction of fermentation inhibiting compounds in lignocellulose hydrolysate

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility. Biotechnol. Bioeng. 2009;102: 1354–1360. © 2008 Wiley Periodicals, Inc.     

Targeted Exosomes for Drug Delivery: Biomanufacturing,Surface Tagging,and Validation

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

Environmental Supply Chain Cooperation and Its Effect on the Circular Economy Practice‐Performance Relationship Among Chinese Manufacturers

Due to resource scarcity and environmental degradation, a new development concept emphasizing environmental concerns, called the circular economy (CE), has been enacted in legislation in China. This environmental management concept can be implemented at three levels, namely, region, industrial zone, and individual enterprise, with the objective of boosting economic development while lessening environmental and resource challenges. Environmental supply chain cooperation (ESCC), an approach that utilizes customer and supplier cooperation in environmental management, has been initiated among Chinese enterprises. Using survey data collected from 396 Chinese manufacturers, we examine the role of ESCC practices in influencing the relationship between implementing CE practices and the achievement of performance outcomes by testing the moderation and mediation effects of ESCC practices on the CE practice‐performance relationship through hierarchical regression analysis. Our data analyses indicate that ESCC practices are useful by moderation and, in some cases, essential by mediation, for Chinese manufacturers seeking to realize the performance targets desired in CE practices. The results highlight the need for Chinese manufacturers to improve supply chain coordination in their implementation of CE. On the policy side, our research findings suggest that ESCC practices are beneficial and, in some cases, necessary for the development of CE in China.