Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash waters and colonization in mice

Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host–microorganism interactions. Here, we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and biofilm formation in leafy-green vegetable wash water. The opgGH mutant strain, which was defective in OPG biosynthesis, initiated the growth at a slower rate in wash waters obtained from spinach, lettuce and green collard and severely impaired biofilm formation. The lack of OPG synthesis did not influence biofilm formation by the opgGH mutant in low-nutrient low-osmolarity laboratory media. In coculture experiments initiated with equal proportions of cells, the opgGH mutant was outnumbered by the wild-type strain under the planktonic as well as the biofilm growth conditions. The opgGH mutant strain poorly colonized mouse organs when introduced orally along with the wild-type strain. This is the first report demonstrating the role of OPGs of Salmonella in competitive colonization of biofilms, planktonic cultures and mouse organs.     

Human exposure to mycotoxins in Egypt

This investigation examined the exposure of Egyptian infants to Aflatoxin M₁ (AfM₁) and of lactating mothers to Aflatoxin B₁, using AfM₁ in human milk as a biomarker for exposure to AfB₁. The presence of ochratoxin A (OA) in human milk was also investigated to determine the levels of infants exposure to OA from human milk. The results indicated that AfM₁ was found in 66 (55 %) of 120 human milk samples with a mean of 0.3 ± 0.53 ng/mL (range 0.02 to 2.09 ng/mL). OA was found in 43 (35.8 %) of 120 human milk samples with a mean of 21.1 ± 13.7 ng/mL (range 5.07 to 45.01 ng/mL), which will cause a daily intake of OA from human milk exceeding the suggested tolerable dose of 5 ng/kg_-1 of OA body weight. On the other side AfM₁ was found in 25 % of blood samples (5 out of 20 samples), at a mean of 1.18 ng/mL, but it was detected only in one urine sample (1 out of 20 samples). OA was detected only in 2 out of 13 blood samples (15.4 %) with an average 3.67 ng/mL. Whereas OA was not detected in all analyzed urine samples.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.     

Predictive motifs derived from cytosine methyltransferases.

Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.     

Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.