A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide.

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

Genetic Manipulation of the Brassicaceae Smut Fungus Thecaphora thlaspeos.

Investigation of plant-microbe interactions greatly benefit from genetically tractable partners to address, molecularly, the virulence and defense mechanisms. The smut fungus Ustilago maydis is a model pathogen in that sense: efficient homologous recombination and a small genome allow targeted modification. On the host side, maize is limiting with regard to rapid genetic alterations. By contrast, the model plant Arabidopsis thaliana is an excellent model with a vast amount of information and techniques as well as genetic resources. Here, we present a transformation protocol for the Brassicaceae smut fungus Thecaphora thlaspeos. Using the well-established methodology of protoplast transformation, we generated the first reporter strains expressing fluorescent proteins to follow mating. As a proof-of-principle for homologous recombination, we deleted the pheromone receptor pra1. As expected, this mutant cannot mate. Further analysis will contribute to our understanding of the role of mating for infection biology in this novel model fungus. From now on, the genetic manipulation of T. thlaspeos, which is able to colonize the model plant A. thaliana, provides us with a pathosystem in which both partners are genetically amenable to study smut infection biology.

Randomized study defining the optimum target interlesion distance in ablation index-guided atrial fibrillation ablation.

AIMS: While the CLOSE protocol proposes a maximally tolerable interlesion distance (ILD) of 6 mm for ablation index ablation index-guided atrial fibrillation (AF) ablation, a target ILD has never been defined. This randomized study sought to establish a target ILD for ablation index-guided AF ablation. METHODS AND RESULTS: Consecutive patients scheduled for first-time pulmonary vein (PV) isolation (PVI) were randomly assigned to ablation protocols with a target ILD of 5.0-6.0 mm or 3.0-4.0 mm, with the primary endpoint of first-pass PVI. In compliance with the CLOSE protocol, the maximum tolerated ILD was 6.0 mm in both study protocols. A target ablation index of ≥550 (anterior) or ≥400 (posterior) was defined for the '5-6 mm' protocol and ≥500 (anterior) or ≥350 (posterior) for the '3-4 mm' protocol. The study was terminated early for superiority of the '3-4 mm' protocol. Forty-two consecutive patients were randomized and 84 ipsilateral PV pairs encircled according to the study protocol. First-pass PVI was accomplished in 35.0% of the '5-6 mm' group and 90.9% of the '3-4 mm' group (P < 0.0001). Median ILD was 5.2 mm in the '5-6 mm' group and 3.6 mm in the '3-4 mm' group (P < 0.0001). In line with the distinct ablation index targets, median ablation index was lower in the '3-4 mm' group (416 vs. 452, P < 0.0001). While mean procedure time was shorter in the '3-4 mm' group (149 ± 27 vs. 167 ± 33min, P = 0.004), fluoroscopy times did not differ significantly (4.7 ± 2.2 vs. 5.1 ± 1.8 min, P = 0.565). CONCLUSION: In ablation index-guided AF ablation, an ILD of 3.0-4.0 mm should be targeted rather than 5.0-6.0 mm. Moreover, the lower target ILD may allow for less extensive ablation at each given point.

Effect of silver nanoparticles and silver ions on growth and adaptive response mechanisms of Pseudomonas putida mt-2.

The distribution and use of nanoparticles increased rapidly during the last years, while the knowledge about mode of action, ecological tolerance and biodegradability of these chemicals is still insufficient. The effect of silver nanoparticles (AgNP) and free silver ions (Ag(+) , AgNO3 ) on Pseudomonas putida mt-2 as one of the best described bacterial strains for stress response were investigated. The effective concentration (EC50) causing 50% growth inhibition for AgNP was about 250 mg L(-1) , whereas this was only 0.175 mg L(-1) for AgNO3 . However, when calculating the amount of free silver ions released from AgNP both tested compounds showed very similar results. Therefore, the antibacterial activity of AgNP can be explained and reduced, respectively, to the amount of silver ions released from the nanoparticles. Both tested compounds showed a strong activation of the unique membrane adaptive response of Pseudomonas strains, the cis-trans isomerization of unsaturated fatty acids, whereas another important adaptive response of these bacteria, changes in cell surface hydrophobicity, measured as water contact angle, was not activated. These results are important informations for the estimation of environmental tolerance of newly developed, active ingredients like silver nanoparticles.

Increased major bleeding risk in patients with kidney dysfunction receiving enoxaparin: a meta-analysis.

PURPOSE: The elimination half-life and consequently the accumulation of enoxaparin increase as glomerular filtration rate (GFR) decreases. A dose adjustment for patients with a GFR <30 ml/min is recommended and considered relatively safe. Our intention was to identify whether the use of enoxaparin is safe and to determine what impact an enoxaparin dose adjustment has on patients with a GFR <60 ml/min. METHODS: A PubMed search and meta-analysis of literature were performed. Studies were analyzed to compare enoxaparin versus other heparins/heparinoids and investigate enoxaparin at different stages of kidney dysfunction. Only controlled trials were considered, and the enoxaparin dose had to be specified. The clinical endpoint was defined as apparent major bleeding. RESULTS: Out of 1,027 publications, 20 studies met the criteria and were analyzed. Our meta-analysis shows that enoxaparin major bleeding complications at a GFR < 60 ml/min increase significantly, with a relative risk (RR) of 1.67 [95% confidence interval (CI) 1.12-2.50) compared with other anticoagulants (p = 0.01). RR for patients on enoxaparin therapy increases exponentially with each stage of chronic kidney disease (CKD stage 1-5) [RR = 0.585 x exp(0.524 x CKD)]. Despite dose adjustment, the major bleeding risk is still significantly increased in patients with a GFR < 60 ml/min versus those with a GFR > 60 ml/min. CONCLUSION: Only patients with a GFR > 60 ml/min can be safely treated with enoxaparin.

Prepubertal urinary estrogen excretion and its relationship with pubertal timing.

Whether prepubertal estrogen production impacts on the timing of puberty is not clear. We aimed to investigate prepubertal 24-h estrogen excretion levels and their association with early and late pubertal markers. Daily urinary excretion rates of estrogens of 132 healthy children, who provided 24-h urine samples 1 and 2 yr before the start of the pubertal growth spurt [age at takeoff (ATO)], were quantified by stable isotope dilution/GC-MS. E-sum3 (estrone + estradiol + estriol) was used as a marker for potentially bioactive estrogen metabolites and E-sum5 (E-sum3 + 16-epiestriol + 16-ketoestradiol) for total estrogen production. Pubertal outcomes were ATO, age at peak height velocity (APHV), duration of pubertal growth acceleration (APHV-ATO), age at Tanner stage 2 for pubic hair (PH2), genital (G2, boys) and breast (B2, girls) development, and age at menarche. Prepubertal urinary estrogen excretions (E-sum3 and E-sum5) were not associated with ATO, APHV, and age at PH2 but with duration of pubertal growth acceleration (P < 0.01) in both sexes. Girls with higher E-sum3 reached B2 0.9 yr (P = 0.04) and menarche 0.3 yr earlier (P = 0.04) than girls with lower E-sum3. E-sum3 was not associated with age at G2 in boys (P = 0.6). For most pubertal variables, the associations with E-sum3 were stronger than with E-sum5. In conclusion, prepubertal estrogens may not be critical for the onset of the pubertal growth spurt but are correlated with its duration in both boys and girls. Prepubertal estrogen levels may already predict the timing of girls' menstruation and breast development but do not appear to affect sexual maturation in boys.

Profiling oestrogens and testosterone in human urine by stable isotope dilution/benchtop gas chromatography-mass spectrometry.

Oestrogens, such as oestrone (E(1)), 17ß-oestradiol (E(2)), oestriol (E(3)) and their biologically active metabolites 2-methoxyoestrone (2-MeOE(1)), 2-hydroxyoestradiol (2-OHE(2)) 16-ketooestradiol (16-OE(2)), 16-epioestriol (16-epiE(3)), as well as testosterone (T) play an important role in physiological and pathological developmental processes during human development. We therefore aimed at developing an isotope dilution/bench top gas chromatography-mass spectrometry (ID/GC-MS) method, based on benchtop GC-MS, for the simultaneous determination ('profiling') of the above analytes in children. The method consisted of equilibration of urine (5 ml) with a cocktail containing stable isotope-labelled analogues of the analytes as internal standards ([2,4-(2)H(2)]E(1), [2,4,16,16-(2)H(4)]E(2), [2,4,17-(2)H(3)]E(3), [16,16,17-(2)H(3)]T, [1,4,16,16-(2)H(4)]2-MeOE(1), [1,4,16,16,17-(2)H(5)]2-OHE(2), [2,4,15,15,17-(2)H(5)]16-OE(2) and [2,4-(2)H(2)]16-epiE(3)). Then, solid-phase extraction (C(18) cartridges), enzymatic hydrolysis (sulphatase from Helix pomatia (type H-1)), re-extraction, purification by anion exchange chromatography and derivatisation to trimethylsilyl ethers followed. The samples were analysed by GC-MS (Agilent GC 6890N/5975MSD; fused silica capillary column 25 m × 0.2 mm i.d., film 0.10 µm). Calibration plots were linear and showed excellent reproducibility with coefficients of determination (r(2)) between 0.999 and 1.000. Intra- and inter-assay coefficients of variation (CV) were <2.21% for all quantified metabolites. Sensitivity was highest for 2-OHE(2) (0.25 pg per absolute injection: signal-to-noise ratio (S/N)=3) and lowest for 16-epiE(3) (2 pg per absolute injection: S/N=2.6), translating into corresponding urine sample analyte concentrations of 0.025 ng ml(-1) and 0.2 ng ml(-1), respectively. Accuracy - determined in a two-level spike experiment - showed relative errors ranging between 0.15% for 16-OE(2) and 11.63% for 2-OHE(2). Chromatography showed clear peak shapes for the components analysed. In summary, we describe a practical, sensitive and specific ID/GC-MS assay capable of profiling the above-mentioned steroids in human urine from childhood onwards.

Functional proteomics using chromophore-assisted laser inactivation.

Proteins are the molecules that fulfil most cellular functions and represent over 90% of drug targets in the market. Chromophore-assisted laser inactivation (CALI) provides a timely and locally restricted protein inactivation and has proven to specifically destroy protein function using dye-coupled ligands and laser irradiation. CALI involves the generation of short-lived radicals thus limiting the radius of covalent modifications to spatially restricted sites on the target molecule. A transient functional inactivation occurs if the radicals modify amino acids of the target protein that are responsible for function. Here we show specific inactivation of several protein targets, that are members of relevant signal transduction pathways. For each of these targets, simple and high throughput screening-scaleable assays have been developed, making it possible to quantify the observed inactivation. Activities of target proteins have been addressed in cell-free as well as cell-based assays employing human primary and tumor-derived cell lines. In all cases, at least 50% inactivation was achieved. The data presented here demonstrate that CALI is a highly versatile tool for validating disease relevant targets at the protein level. This approach also takes into account post-translational modifications like phosphorylation, glycosylation or acylation, thereby enlarging its applicability for many different types of targets.