New amino acid residue type identification experiments valid for protonated and deuterated proteins

Two experiments are presented that yield amino acid type identification of individual residues in a protein by editing the ¹H?C¹⁵N correlations into four different 2D subspectra, each corresponding to a different amino acid type class, and that can be applied to deuterated proteins. One experiment provides information on the amino acid type of the residue preceding the detected amide ¹H?C¹⁵N correlation, while the other gives information on the type of its own residue. Versions for protonated proteins are also presented, and in this case it is possible to classify the residues into six different classes. Both sequential and intraresidue experiments provide highly complementary information, greatly facilitating the assignment of protein resonances. The experiments will also assist in transferring the assignment of a protein to the spectra obtained under different experimental conditions (e.g. temperature, pH, presence of ligands, cofactors, etc.).     

Effect of Mg, Si, and Sr on growth and antioxidant activity of the marine microalga Tetraselmis suecica

Efforts to increase the productivity of microalgal cultures have been focused on the improvement of photobioreactors, but little attention has been paid to the nutritional requirements of microalgae in order to improve culture media formulation. In this study, the main goal was obtaining a high productivity for Tetraselmis suecica (Chlorophyta) in semicontinuous culture by adding magnesium (Mg), silicon (Si), and strontium (Sr) at concentrations from 0.01 to 10 mM; at the time, the effect on steady-state cell density, biochemical composition, and antioxidant activity of T. suecica was evaluated. Because productivity is higher in high-density cultures, the work was focused many times to cell density. Mg (3 mM) and Sr (0.1 mM) added separately reached the highest steady-state cell density (7.0?×?10⁶?±?0.4 cells mL^?1) in comparison to control (4.2?±?0.1 cells mL^?1), but simultaneous addition had a synergic effect, achieving 8.7?×?10⁶?±?0.6 cells mL^?1. Silicon (3 mM) significantly affected the steady-state cell density, reaching 6.0?±?0.3 cells mL^?1 and increased the cell ash-free dry weight, reaching 127?±?7.9 pg cell^?1 in comparison to control (102.7?±?5.0 pg cell^?1), resulting in an ash-free dry weight productivity of 0.75?±?0.07 g?L^?1 day^?1. The highest fatty acids content and antioxidant activity, measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method were obtained with Sr 10 mM. Sr treatments showed a high correlation (R ²?=?0.98) between DPPH inhibition and polyphenolic content, explaining its high antioxidant activity. Therefore, the addition of Mg, Si, and Sr to culture medium of T. suecica is recommended to achieve high steady-state cell density in semicontinuous cultures.     

On the use of possibility theory in uncertainty analysis of life cycle inventory

Purpose  

The purpose of this paper is to enhance the mathematical and physical understanding of practitioners of uncertainty analysis of life cycle inventory (LCI), on the application of possibility theory. The main questions dealt with are (1) clear definition of the terms—“necessity–possibility,” “probability,” “belief–plausibility,” and of their mutual relationships; (2) what justifies the substitution of classical probability for possibility; (3) mutual comparison of, and transformations in both senses between probability and possibility uncertainty measures; (4) how to construct meaningful input possibility measures from available probabilistic/statistic information; and (5) comparative analysis of the solutions of the problem of data uncertainty propagation in LCI, afforded, respectively, by probabilistic Monte Carlo simulation and possibilistic fuzzy interval arithmetic.     

The missing link in coenzyme A biosynthesis: PanM (formerly YhhK), a yeast GCN5 acetyltransferase homologue triggers aspartate decarboxylase (PanD) maturation in Salmonella enterica

Coenzyme A (CoA) is an essential cofactor for all forms of life. The biochemistry underpinning the assembly of CoA in Escherichia coli and other enterobacteria is well understood, except for the events leading to maturation of the L-aspartate-α-decarboxylase (PanD) enzyme that converts pantothenate to β-alanine. PanD is synthesized as pro-PanD, which undergoes an auto-proteolytic cleavage at residue Ser25 to yield the catalytic pyruvoyl moiety of the enzyme. Since 1990, it has been known that E. coli yhhK strains are pantothenate auxotrophs, but the role of YhhK in pantothenate biosynthesis remained an enigma. Here we show that Salmonella enterica yhhK strains are also pantothenate auxotrophs. In vivo and in vitro evidence shows that YhhK interacts directly with PanD, and that such interactions accelerate pro-PanD maturation. We also show that S. enterica yhhK strains accumulate pro-PanD, and that not all pro-PanD proteins require YhhK for maturation. For example, the Corynebacterium glutamicum panD(+) gene corrected the pantothenate auxotrophy of a S. enterica yhhK strain, supporting in vitro evidence obtained by others that some pro-PanD proteins autocleave at faster rates. We propose the name PanM for YhhK to reflect its role as a trigger of pro-PanD maturation by stabilizing pro-PanD in an autocleavage-prone conformation.     

Aclidinium bromide, a novel long-acting muscarinic antagonist for COPD with improved preclinical renal and urinary safety profile

AimsAclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium.Main methodsThe effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10–1000 μg/kg, s.c.), anaesthetised Beagle dogs (1000 μg/kg, i.v.) and anaesthetised guinea pigs (3–100 μg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only).Key findingsAclidinium 1000 μg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 μg/kg which significantly decreased this parameter (p < 0.05). Aclidinium 1000 μg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3–100 μg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 μg/kg decreased the peak micturition pressure (p < 0.05), increased the volume of urine retained in the bladder (p < 0.01) and showed a trend to decrease the volume of urine excreted.SignificanceAclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.     

Grazing-induced effects on soil properties modify plant competitive interactions in semi-natural mountain grasslands

Plant-soil feedbacks are widely recognized as playing a significant role in structuring plant communities through their effects on plant-plant interactions. However, the question of whether plant-soil feedbacks can be indirectly driven by other ecological agents, such as large herbivores, which are known to strongly modify plant community structure and soil properties, remains poorly explored. We tested in a glasshouse experiment how changes in soil properties resulting from long-term sheep grazing affect competitive interactions (intra- and inter-specific) of two graminoid species: Nardus stricta, which is typically abundant under high sheep grazing pressure in British mountain grasslands; and Eriophorum vaginatum, whose abundance is typically diminished under grazing. Both species were grown in monocultures and mixtures at different densities in soils taken from adjacent grazed and ungrazed mountain grassland in the Yorkshire Dales, northern England. Nardus stricta performed better (shoot and root biomass) when grown in grazing-conditioned soil, independent of whether or not it grew under inter-specific competition. Eriophorum vaginatum also grew better when planted in soil from the grazed site, but this occurred only when it did not experience inter-specific competition with N. stricta. This indicates that plant-soil feedback for E. vaginatum is dependent on the presence of an inter-specific competitor. A yield density model showed that indirect effects of grazing increased the intensity of intra-specific competition in both species in comparison with ungrazed-conditioned soil. However, indirect effects of grazing on the intensity of inter-specific competition were species-specific favouring N. stricta. We explain these asymmetric grazing-induced effects on competition on the basis of traits of the superior competitor and grazing effects on soil nutrients. Finally, we discuss the relevance of our findings for plant community dynamics in grazed, semi-natural grasslands.     

Harnessing the rhizosphere microbiome through plant breeding and agricultural management

Background

The need to enhance the sustainability of intensive agricultural systems is widely recognized One promising approach is to encourage beneficial services provided by soil microorganisms to decrease the inputs of fertilizers and pesticides. However, limited success of this approach in field applications raises questions as to how this might be best accomplished.

Scope

We highlight connections between root exudates and the rhizosphere microbiome, and discuss the possibility of using plant exudation characteristics to selectively enhance beneficial microbial activities and microbiome characteristics. Gaps in our understanding and areas of research that are vital to our ability to more fully exploit the soil microbiome for agroecosystem productivity and sustainability are also discussed.

Conclusion

This article outlines strategies for more effectively exploiting beneficial microbial services on agricultural systems, and cals attention to topics that require additional research.     

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.