Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

Background  

We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity.     

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.     

The rate of bone mineralization in birds is directly related to alkaline phosphatase activity

Recent studies have suggested that a biochemical marker, plasma alkaline phosphatase (ALP), can be used as a general indicator of skeletal development in vertebrate animals. In birds, age-related variation in ALP activity, presumably due to bone formation processes, has been demonstrated, but to date, a direct connection between bone mineralization and enzyme activity has been elusive. In this study, we show that the activity of a bone isoform of ALP (bone ALP) is closely related to the overall rate of skeletal mineralization in nestlings of a small passerine bird, the great tit (Parus major L). Moreover, bone ALP activity predicted the rate of mineralization of leg and wing bones but not that of the skull. Liver isoform of ALP was only marginally related to the overall rate of skeletal mineralization, while no association with the mineralization of long bones was found. We conclude that bone ALP activity in the blood plasma is a reliable biomarker for skeletal mineralization in birds. This marker enables detection of subtle developmental differences between chicks of similar structural size, potentially facilitating the prediction of offspring mid- and long-term survival.     

Synergistic cellulose hydrolysis can be described in terms of fractal-like kinetics

A fractal-like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme-to-substrate ratios showed an apparent substrate inhibition consistent with a case where two-dimensional (2-D) surface diffusion of the cellobiohydrolase is rate-limiting. The action of Cel7A core and Cel5A was instead consistent with a three-dimensional (3-D) diffusion-based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme-to-substrate ratio, but this effect was gradually reduced at lower enzyme-to-substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal-like kinetics analogue of a first-order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions.     

Effects of light intensity and duration on leaf hydraulic conductance and distribution of resistance in shoots of silver birch (Betula pendula)

Variation in leaf hydraulic conductance (K(L)) and distribution of resistance in response to light intensity and duration were examined in shoots of silver birch (Betula pendula Roth). K(L) was determined on detached shoots using the evaporative flux method (transpiration was measured with a porometer and water potential drop with a pressure chamber). Although K(L) depended on light duration per se, its dynamics was largely determined by leaf temperature (T(L)). Both upper-crown leaves and branches developed in well-illuminated environment exhibited higher hydraulic efficiency compared with the lower crown, accounting for vertical trends of apparent soil-to-leaf hydraulic conductance in canopy of silver birch revealed in our previous studies. K(L) varied significantly with light intensity, the highest values for both shade and sun foliage were recorded at photosynthetic photon flux density of 330 micromol m(-2) s(-1). Light responses of K(L) were associated evidently with an irradiance-mediated effect on extravascular tissues involving regulation of cell membrane aquaporins. Effects of irradiance on K(L) resulted in changes of Psi(L), bringing about considerable alteration in partitioning of the resistance between leaves and branch (leafless shoot stem): the contribution of leaves to the shoot total resistance decreased from 94% at -1.0 MPa to 75% at -0.2 MPa. Treatment with HgCl2 decreased hydraulic conductance of both leaves and branches, implying that condition of bordered pit membranes or shoot living tissues may be involved in responses of xylem conductance to Hg2+.     

Physical Activity and Bone Mineral Accrual in Boys with Different Body Mass Parameters during Puberty: A Longitudinal Study

The aim of our longitudinal study was to investigate the relationships between physical activity and bone mass in boys with different body mass status during the years surrounding pubertal growth spurt. Two hundred and six boys entering puberty took part in this study. The subjects were divided into underweight (), normal weight (), overweight () and obese () groups at baseline according to age related categories. Whole-body DXA scans were performed at baseline, after 12 and 24 months to assess body composition (lean body mass, fat mass), and total body (TB), lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) parameters. Physical activity was measured by 7- day accelerometry. For longitudinal analysis, multilevel fixed effects regression models were constructed. Biological age, height and lean body mass had an effect for explanation of TB BMD, FN BMD and LS BMD. Moderate to vigorous physical activity (MVPA), vigorous physical activity (VPA) and sedentary time (SED) had the significant effect only on FN BMD. Being an underweight boy at the baseline indicated greater chance (p<0.01) to have lower TB BMD in the future (2 years at follow up) development, compared to normal weight (estimates = −0.038), overweight (estimates = −0.061) and obese boys (estimates = −0.106).     

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency (kcat/Km(H2O2)) of the cellulose peroxygenase reaction (k_cat = 8.5 s⁻¹, and Km(H2O2)=30μM) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H₂O₂ in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.     

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N?H? and NH?OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L?1 of each NH?OH and N?H?, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO??. Various combinations of N?H?, NH?OH, NH??, and NO?? were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N?H? concentration (4.38 mg N L?1) present in these batches was 5.43 mg N g?1 TSS h?1, whereas equimolar concentrations of N?H? and NH?OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.