High prevalence of VanA-type vancomycin-resistant Enterococci in Austrian poultry

Fecal samples from humans and food-producing animals were analyzed for the presence of vancomycin-resistant enterococci (VRE). The VRE carriage rate in humans was 6%, and there was a predominance of VanC-type resistance. Enterococcus faecium with vanA-mediated resistance was frequent in broiler chickens (42%) but rare in cattle and pig samples.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.     

Prevention of beta strand movement into a zymogen-like position does not confer higher activity to coagulation factor VIIa

Coagulation factor VIIa (FVIIa) belongs to the chymotrypsin family of S1 peptidases, whose members require the cleavage of at least one internal peptide bond to attain an active conformation. FVIIa also requires association with tissue factor (TF) to attain full catalytic competency. Without this, FVIIa has very low activity toward peptide and physiologic substrates. Reregistration of beta strands has been suggested to play a part in the activation of FVII, and their positioning is possibly important for the active conformation of FVIIa. To scrutinize this hypothesis, we have designed FVIIa variants which prevent beta strand movement and lock FVIIa in the alleged active conformation. The V299M mutation, alone or combined with the L280I mutation, was introduced to alter the first of two Leu-X-Val motifs in beta strand B2 and thereby prevent reregistration. Along the same line, C164V/V299C-FVIIa has a new disulfide which would keep beta strand B2 in the registration of active FVIIa. The amidolytic and proteolytic activities of V299M-, L280I/V299M-, and C164V/V299C-FVIIa were indistinguishable from or lower than those of wild-type FVIIa, and none of the mutants displayed an altered exposure of the N-terminal amino group of the protease domain. Moreover, the affinities of mutant and native FVIIa for TF increased to a similar extent upon incorporation of an active site inhibitor, and the enzymatic activities were equally stimulated by TF. In conclusion, we found no evidence that the mutants were in a more active state than native FVIIa. Thus, the proposed beta strand reregistration, if part of the regulatory mechanism governing FVIIa activity, apparently does not suffice for the transformation of FVIIa into an enzymatically active conformation. Our data raise the possibility that the structural differences between enzymatically latent (zymogen-like) and active FVIIa resemble those between trypsinogen and trypsin.     

Fluctuating asymmetry,leaf thickness and herbivory in <Emphasis Type="Italic">Tibouchina granulosa</Emphasis>: an altitudinal gradient analysis

Gradient of stressful conditions affect plant physiological and morphological traits. Previous studies have shown that plants located at higher altitudes might exhibit higher levels of both fluctuating asymmetry and leaf thickness. Although it is expected that higher fluctuating asymmetry levels should be accompanied by higher leaf consumption by herbivores, lower herbivory could be expected for elevated leaf thickness. Aiming to investigate this contradiction our objective was to determine the effects of altitude on fluctuating asymmetry and leaf thickness, and evaluate the importance of these two morphological traits on herbivory levels of Tibouchina granulosa Cogn. (Melastomatecea) in Brazilian Atlantic Forest. The study was conducted in southern Brazil, along a continuous altitudinal gradient raging from 1275 to 1950 m, where we measured fluctuating asymmetry, leaf thickness and herbivory from leaves of 29 individuals of T. granulosa. There was a positive effect of altitude on both fluctuating asymmetry and leaf thickness but only fluctuating asymmetry was related to herbivore. Our results suggest that as altitude increases plants face more stressful conditions, leading to higher fluctuating asymmetry. This may lead to a higher nutritional quality of leaves and herbivores may use leaf asymmetry as a cue for plant quality. The lack of a relationship between leaf thickness and herbivory gives us evidence that, in the studied location, leaf thickness is not primarily used as plant defense and probably has other functions related, for example, to water, solar radiation, and nutrient stresses. These results may be considered a baseline for the understanding on how altitudinal stress and potential herbivory pressure influence plant populations.     

Structural, mechanical and electronic properties of nano-fibriform silica and its organic functionalization by dimethyl silane: a SCC-DFTB approach

Self-consistent-charge density-functional tight-binding (SCC-DFTB) approximated method was employed to investigate the structural, mechanical and electronic properties of the zigzag and armchair nano-fibriform silica (SNTs) and their outer surface organic modified derivatives (MSNTs) with internal radii in the range of 8 to 36 Å. The strain energy curves showed that the nanotubes structures are energetically more stable compared to the respective sheet structures. External hydroxyl dihedral angles in silica nanotubes have small influence, about 0.5 meV.atom^?1, in the strain energy curve tendency of those materials favoring the zigzag chirality. The chemical modification of outer surface of SNTs by dimethyl silane group affects their relative stability favoring the armchair chirality in approximately 2 meV.atom^?1. MSNTs have axial elastic constants, Young’s moduli, determined at the harmonic approximation, around 100 GPa smaller than the respective SNTs. The Young’s moduli of zigzag and armchair SNTs are in the range of 150–195 GPa and 232–260 GPa, respectively. And for the zigzag and armchair MSNTs these values are in the range of 77–89 and 110–140 GPa, respectively. The SNTs and MSNTs were characterized as insulators with band gaps around 8–10 eV.

Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.     

Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.