Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications

Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.     

Crystallization and preliminary X-ray diffraction study of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea

1,3,8-Trihydroxynaphthalene reductase was crystallized in the presence of NADPH and the inhibitor tricyclazole. The crystals are trigonal, space group P3₁21 or its enantiomorph P3₂21. Two crystal forms with slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b = 142.6 Å, c = 70.1 Å and form B cell dimensions a = b = 142.6 Å, c = 72.9 Å. The diffraction pattern of the latter crystal form extends to 2.5 Å resolution.     

Relative importance of competition and plant–soil feedback,their synergy,context dependency and implications for coexistence

Plants interact simultaneously with each other and with soil biota, yet the relative importance of competition vs. plant–soil feedback (PSF) on plant performance is poorly understood. Using a meta‐analysis of 38 published studies and 150 plant species, we show that effects of interspecific competition (either growing plants with a competitor or singly, or comparing inter‐ vs. intraspecific competition) and PSF (comparing home vs. away soil, live vs. sterile soil, or control vs. fungicide‐treated soil) depended on treatments but were predominantly negative, broadly comparable in magnitude, and additive or synergistic. Stronger competitors experienced more negative PSF than weaker competitors when controlling for density (inter‐ to intraspecific competition), suggesting that PSF could prevent competitive dominance and promote coexistence. When competition was measured against plants growing singly, the strength of competition overwhelmed PSF, indicating that the relative importance of PSF may depend not only on neighbour identity but also density. We evaluate how competition and PSFs might interact across resource gradients; PSF will likely strengthen competitive interactions in high resource environments and enhance facilitative interactions in low‐resource environments. Finally, we provide a framework for filling key knowledge gaps and advancing our understanding of how these biotic interactions influence community structure.     

Modelling biological cell attachment and growth on adherent surfaces

A mathematical model, in the form of an integro-partial differential equation, is presented to describe the dynamics of cells being deposited, attaching and growing in the form of a monolayer across an adherent surface. The model takes into account that the cells suspended in the media used for the seeding have a distribution of sizes, and that the attachment of cells restricts further deposition by fragmenting the parts of the domain unoccupied by cells. Once attached the cells are assumed to be able to grow and proliferate over the domain by a process of infilling of the interstitial gaps; it is shown that without cell proliferation there is a slow build up of the monolayer but if the surface is conducive to cell spreading and proliferation then complete coverage of the domain by the monolayer can be achieved more rapidly. Analytical solutions of the model equations are obtained for special cases, and numerical solutions are presented for parameter values derived from experiments of rat mesenchymal stromal cells seeded onto thin layers of collagen-coated polyethylene terephthalate electrospun fibers. The model represents a new approach to describing the deposition, attachment and growth of cells over adherent surfaces, and should prove useful for studying the dynamics of the seeding of biomaterials.     

Sensitivity and Acclimation of Three Canopy-Forming Seaweeds to UVB Radiation and Warming

Canopy-forming seaweeds, as primary producers and foundation species, provide key ecological services. Their responses to multiple stressors associated with climate change could therefore have important knock-on effects on the functioning of coastal ecosystems. We examined interactive effects of UVB radiation and warming on juveniles of three habitat-forming subtidal seaweeds from Western Australia–Ecklonia radiata, Scytothalia dorycarpa and Sargassum sp. Fronds were incubated for 14 days at 16–30°C with or without UVB radiation and growth, health status, photosynthetic performance, and light absorbance measured. Furthermore, we used empirical models from the metabolic theory of ecology to evaluate the sensitivity of these important seaweeds to ocean warming. Results indicated that responses to UVB and warming were species specific, with Sargassum showing highest tolerance to a broad range of temperatures. Scytothalia was most sensitive to elevated temperature based on the reduced maximum quantum yields of PSII; however, Ecklonia was most sensitive, according to the comparison of activation energy calculated from Arrhenius’ model. UVB radiation caused reduction in the growth, physiological responses and thallus health in all three species. Our findings indicate that Scytothalia was capable of acclimating in response to UVB and increasing its light absorption efficiency in the UV bands, probably by up-regulating synthesis of photoprotective compounds. The other two species did not acclimate over the two weeks of exposure to UVB. Overall, UVB and warming would severely inhibit the growth and photosynthesis of these canopy-forming seaweeds and decrease their coverage. Differences in the sensitivity and acclimation of major seaweed species to temperature and UVB may alter the balance between species in future seaweed communities under climate change.     

Ageing Cattle: The Use of Radiographic Examinations on Cattle Metapodials from Eketorp Ringfort on the Island of ?land in Sweden

In this paper conventional X-ray analysis of cattle metapodials is used to study the age structure of slaughtered cattle at Eketorp ringfort on the island of Öland, Sweden. The X-ray analyses suggest that several animals in both phases were slaughtered aged 4–8 years. More oxen/bulls than cows reached the advanced age of over 8 years, yet in phase III more oxen/bulls seem to have been slaughtered between the ages of 2 and 8 years. These differences may reflect a change in demand for meat related to the character of the site. The results also show a correlation between metapodials with a pathology connected to biomechanical stress and older animals. This suggests that male cattle were used both in meat production and as draught animals. Asymmetry in male metatarsals such as distal broadening of the lateral part of the medial trochlea was visible on the X-ray images. The bone element also indicates a denser outer cortex of the medial diaphysis in comparison to the inner medulla. This could be the result of repetitive mechanical stress. Two metatarsals from cows were documented with distal asymmetry indicating that cows were also used as working animals. Bone elements with changes in the articular surfaces were more common in metapodials from cows with an X-ray age of over 3–4 years. These results highlighted the slaughter age difference between oxen/bulls and cows, enabling a better understanding of animal husbandry and the selection of draught cattle at Eketorp ringfort.     

Correspondence of ectomycorrhizal diversity and colonisation of willows (Salix spp.) grown in short rotation coppice on arable sites and adjacent natural stands

Willows (Salix spp.) are mycorrhizal tree species sometimes cultivated as short rotation coppice (SRC) on arable sites for energy purposes; they are also among the earliest plants colonising primary successional sites in natural stands. The objective of this study was to analyse the degree of colonisation and diversity of ectomycorrhizal (EM) communities on willows grown as SRC in arable soils and their adjacent natural or naturalized stands. Arable sites usually lack ectomycorrhizal host plants before the establishment of SRC, and adjacent natural or naturalized willow stands were hypothesized to be a leading source of ectomycorrhizal inoculum for the SRC. Three test sites including SRC stands (Salix viminalis, Salix dasyclados, and Salix schwerinii) and adjacent natural or naturalized (Salix caprea, Salix fragilis, and Salix?×?mollissima) stands in central Sweden were investigated on EM colonisation and morphotypes, and the fungal partners of 36 of the total 49 EM fungi morphotypes were identified using molecular tools. The frequency of mycorrhizas in the natural/naturalized stands was higher (two sites) or lower (one site) than in the corresponding cultivated stands. Correspondence analysis revealed that some EM taxa (e.g. Agaricales) were mostly associated with cultivated willows, while others (e.g. Thelephorales) were mostly found in natural/naturalized stands. In conclusion, we found strong effects of sites and willow genotype on EM fungi formation, but poor correspondence between the EM fungi abundance and diversity in SRC and their adjacent natural/naturalized stands. The underlying mechanism might be selective promotion of some EM fungi species by more effective spore dispersal.     

Discovery and characterization of AZD9272 and AZD6538—Two novel mGluR5 negative allosteric modulators selected for clinical development

AZD9272 and AZD6538 are two novel mGluR5 negative allosteric modulators selected for further clinical development. An initial high-throughput screening revealed leads with promising profiles, which were further optimized by minor, yet indispensable, structural modifications to bring forth these drug candidates. Advantageously, both compounds may be synthesized in as little as one step. Both are highly potent and selective for the human as well as the rat mGluR5 where they interact at the same binding site than MPEP. They are orally available, allow for long interval administration due to a high metabolic stability and long half-lives in rats and permeate the blood brain barrier to a high extent. AZD9272 has progressed into phase I clinical studies.     

Phenotypic and genetic differentiation among yellow monkeyflower populations from thermal and non-thermal soils in Yellowstone National Park

In flowering plants, soil heterogeneity can generate divergent natural selection over fine spatial scales, and thus promote local adaptation in the absence of geographic barriers to gene flow. Here, we investigate phenotypic and genetic differentiation in one of the few flowering plants that thrives in both geothermal and non-thermal soils in Yellowstone National Park (YNP). Yellow monkeyflowers (Mimulus guttatus) growing at two geothermal ("thermal") sites in YNP were distinct in growth form and phenology from paired populations growing nearby (<500?m distant) in non-thermal soils. In simulated thermal and non-thermal environments, thermal plants remained significantly divergent from non-thermal plants in vegetative, floral, mating system, and phenological traits. Plants from both thermal populations flowered closer to the ground, allocated relatively more to sexual reproduction, were more likely to initiate flowering under short daylengths, and made smaller flowers that could efficiently self-fertilize without pollinators. These shared differences are consistent with local adaptation to life in the ephemeral window for growth and reproduction created by winter and spring snowmelt on hot soils. In contrast, habitat type (thermal vs. non-thermal) explained little of the genetic variation at neutral markers. Instead, we found that one thermal population (Agrostis Headquarters; AHQ-T) was strongly differentiated from all other populations (all F (ST)?>?0.34), which were only weakly differentiated from each other (all F (ST)?相似文献