Development of a microplate-based scintillation proximity assay for MraY using a modified substrate.

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

Nonlinear model for Marangoni convection.

We have constructed a Lorenz-like model for Marangoni convection with finite wave number in large aspect ratio situations. Within the model, there is exchange of stabilities at the onset of convection and beyond the onset there is onset of oscillations due to the presence of surface fluctuations. The oscillations become chaotic as the Marangoni number is increased.

Rayleigh-Marangoni convection in a critical fluid: a tale of two crossovers.

We show that if we take a thin layer of fluid where surface tension effects are supposed to dominate and gradually bring the mean temperature of the layer closer and closer to the liquid vapor critical point, then first there is a crossover from Marangoni to Rayleigh-Benard convection and thence to a convection whose onset is determined by the Schwarzchild criterion.

Standing orientation and behaviour of goats during short-haul road transportation.

A field study was conducted to observe the standing orientation and behaviour among a group of meat type goats during transit by road. Randomly selected goats were observed from 10 runs (10 goats per run) carried out on the same road and the duration of each run was about 50min. Two observers stood on the deck of the truck and each noted the ongoing activities of five animals. The most frequent standing orientation adopted by goats was parallel to the truck's direction of travel (24.7+/-2.1min) followed by diagonal orientation (11.9+/-1.6min) and perpendicular orientation (9.1+/-1.4min) during the 50min road journey. The goats were not observed to orient themselves opposite to the truck's direction of travel. Although, the animals changed their orientation frequently, apparently to maintain balance, there was a clear bias against the perpendicular and diagonal orientations. Mean frequencies for the various behaviours were: bleating (1.3), jumping (0.7), pushing (0.9), fighting (0.7), falling (3.1), urination (0.5), defecation (0.3) and rumination (0.1). Jumping and bleating mostly occurred at the start of the journey.Virtually all fallings were associated with the driver's changing speed of vehicle by braking (43.4%), cornering (32.4%), speed bump (12.3%) and acceleration (11.9%). These falls, which hamper the well being of animals in transit and are also responsible for bruising which can be prevented by careful driving and by adjusting the speed of the vehicle gradually.

Evaluation of blood oxidative stress-related parameters in alcoholic liver disease and non-alcoholic fatty liver disease.

Oxidative stress is implicated in the pathogenesis of liver disease. We investigated oxidative stress-related parameters and correlated with clinical findings in 35 non-alcoholic fatty liver disease (NAFLD) patients, 38 alcoholic liver disease (ALD) patients and 38 normal subjects. NAFLD patients showed significantly higher body mass index, cholesterol, LDL-cholesterol, VLDL-cholesterol levels and transaminase activities compared to the other two groups. Haematological parameters were significantly altered in ALD patients and were reported only in male subjects. Glutathione content, catalase activity, glutathione reductase activity and glutathione peroxidase activity in NAFLD patients were reduced by 10.7 %, 18.5 %, 8.1 % and 16.8 %, respectively, and in ALD patients by 21.8 %, 29.6 %, 24.3 % and 45.3 %, respectively, compared to the normal group. However, thiobarbituric acid reactive substance content, superoxide dismutase activity and glutathione s-transferase activity were increased by 35.2 %, 31.6 % and 5.4 %, respectively, in NAFLD patients, and in ALD patients by 75.2 %, 72.7 % and 32.4 %, respectively, compared to the normal group. Oxidative stress is associated with collagen production and leads to fibrosis. Type IV collagen level in NAFLD patients (190.6 +/- 83 ng/mL) was significantly higher than in the normal group (124.5 +/- 14.5 ng/mL) and lower than in ALD patients (373.4 +/- 170 ng/mL). While type IV collagen level of >124 ng/mL was a predictor of NAFLD patients from normal subjects, elevated ALT (>40 IU/L) activity could discriminate either of the liver disease patients from normal subjects.

Marangoni convection on an inhomogeneous substrate.

We consider an inhomogeneous substrate for Marangoni convection. The inhomogeneity shows up in a nonuniform temperature distribution which we model by a periodic variation. The response can exhibit parametric resonance. Both resonant and nonresonant responses are considered and a possible strong effect on wave number selection indicated.

Transfection and recombination with molecularly cloned derivatives of avian sarcoma virus UR2.

A cloned version of avian sarcoma virus UR2, plasmid pKD6, which includes the full, nonpermuted proviral sequence between two LTR regions, has been prepared. The plasmid is biologically active in transfection experiments, even when intact. Two transformation-defective mutants with nonoverlapping deletions within the transforming gene ros were constructed from pKD6. These mutants recombine to produce transforming virus when mixed DNA from both is used to transfect chick embryo fibroblasts along with helper virus DNA. However, recombination was not readily detected when cells were coinfected with fluids harvested from cultures separately transfected with DNA from each mutant. This, and marker rescue experiments with a temperature-sensitive mutant of UR2 defective in transformation but able to replicate, suggest that deletion mutants of UR2 do not propagate efficiently.

Magnetic resonance venography in assessing venous patency after multiple venous catheters.

BACKGROUND/PURPOSE: Venous thrombosis is a well-recognised complication of central venous catheters (CVC). The aim of the study was to assess the value of magnetic resonance venography (MRV) in assessing venous patency in children with suspected venous thrombosis. METHODS: Contrast studies through the CVC (linogram) and Doppler ultrasonography were the initial investigations performed in children with suspected CVC-related thrombosis. Two-dimensional gated inflow and phase contrast MRV also was performed to assess the extent of venous thrombosis and to locate patent veins for replacement CVC. When the MRV identified a suitable patent vein, the CVC was reinserted by direct venous cut down or the percutaneous method under a general anaesthetic. RESULTS: A total of 25 children (median age, 5 years; range, 2 months to 17 years) who had multiple CVC insertions (median, 3; range, 1-9), underwent MRV for suspected venous thrombosis. Of 10 patients in whom the catheter was completely occluded, MRV identified extensive thrombosis of the central veins in 6. In 7 other children the linogram showed adherent thrombus at the tip of the CVC only. In 5 of these 7 children MRV showed extensive thrombosis of the vein in which the catheters were placed. Doppler ultrasonography diagnosed thrombotic occlusion of the neck veins in 7 children. The MRV studies showed more extensive thrombosis in 4 of these 7 patients. Additionally, MRV showed thrombosis of the intrathoracic veins in 11 patients who had patent neck veins on ultrasound scan. MRV identified a patent vein for reinsertion of CVC in 22 of 25 children. At operation, venous patency was confirmed in 20 patients (91%). CONCLUSION: MRV in children with suspected CVC-related thrombosis is more accurate than Doppler ultrasonography, and contrast studies for defining the extent of venous thrombosis. MRV correctly shows venous anatomy and patency for reinsertion of CVC.