Aldehyde-Thionin: A Stain Having Similar Properties to Aldehyde-Fuchsin

A mixture containing thionin, 0.5 gm; paraldehyde, 7.5 ml; concentrated HCl, 1 ml; and 70% ethanol, 91.5 ml, when allowed to ripen for several days, produces a stain which, when applied to sections of tissue fixed in a Zenker-based fixative, resembles in its effects the aldehyde-fuchsin stain of Gomori, but presents certain advantages.     

Rat liver alcohol dehydrogenase. Purification and properties

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria,Cylindrospermopsis and Microcystis

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.     

Some problems associated with determining iron in plants

Summary 1. Iron which is shown to be present as a contaminant on leaf surfaces of tomato can be removed completely by washing the leaves for 30 sec in 0.3% teepol and water followed by a 30 sec rinsing in distilled water.2. By using radioactive iron it is shown that there was no loss of the metal from within the plants during leaf washing. Agreement between chemical and radioassay data for iron showed that the metal was completely removed by the wash procedure.3. In tomato grown in the glasshouse consistent differences between the iron contents of normal and chlorotic leaves were obtained after leaf washing. When the plants were grown in a dustproof chamber differences in iron status of green and chlorotic leaves were obtained without washing.4. Added radioactive and carrier iron was fully recovered from acid digests of a variety of plant material, specially chosen to represent a range of silica contents.     

Behaviour of iron chelates in calcareous soils

Summary A highly calcareous clay soil was treated with six iron chelates, those formed by RA 155, RA 156, RA 157, RA 159, ATA or HEG, and then extracted with an aqueous solution after intervals of up to 30 days.About 80% of total RA 155 was recovered at each sampling time, but after 15 days approximately one-third of the iron in it had been replaced by calcium. Some of the Fe-RA 156 and Fe-RA 159 was sorbed by the clay, but no calcium was found to have replaced iron chelated with RA 156, RA 157 or RA 159.The four chelates of the RA series were labelled with Fe⁵⁹. The amount of isotopic exchange taking place between the Fe⁵⁹ and soil iron was found to be negligible.A very low percentage of Fe-ATA and Fe-HEG was recovered from the soil. Approximately 50% of the Fe-ATA was sorbed by the clay, and in the remainder the chelated iron was replaced by calcium. The cause of the loss of Fe-HEG could not be determined.     

Routine antenatal anti-D prophylaxis in women who are Rh(D) negative: meta-analyses adjusted for differences in study design and quality

Background

To estimate the effectiveness of routine antenatal anti-D prophylaxis for preventing sensitisation in pregnant Rhesus negative women, and to explore whether this depends on the treatment regimen adopted.

Methods

Ten studies identified in a previous systematic literature search were included. Potential sources of bias were systematically identified using bias checklists, and their impact and uncertainty were quantified using expert opinion. Study results were adjusted for biases and combined, first in a random-effects meta-analysis and then in a random-effects meta-regression analysis.

Results

In a conventional meta-analysis, the pooled odds ratio for sensitisation was estimated as 0.25 (95% CI 0.18, 0.36), comparing routine antenatal anti-D prophylaxis to control, with some heterogeneity (I ² = 19%). However, this naïve analysis ignores substantial differences in study quality and design. After adjusting for these, the pooled odds ratio for sensitisation was estimated as 0.31 (95% CI 0.17, 0.56), with no evidence of heterogeneity (I ² = 0%). A meta-regression analysis was performed, which used the data available from the ten anti-D prophylaxis studies to inform us about the relative effectiveness of three licensed treatments. This gave an 83% probability that a dose of 1250 IU at 28 and 34 weeks is most effective and a 76% probability that a single dose of 1500 IU at 28–30 weeks is least effective.

Conclusion

There is strong evidence for the effectiveness of routine antenatal anti-D prophylaxis for prevention of sensitisation, in support of the policy of offering routine prophylaxis to all non-sensitised pregnant Rhesus negative women. All three licensed dose regimens are expected to be effective.     

Bacterial community composition of a wastewater treatment system reliant on N2 fixation

The temporal stability and change of the dominant phylogenetic groups of the domain bacteria were studied in a model plant-based industrial wastewater treatment system showing high levels of organic carbon removal supported by high levels of N₂ fixation. Community profiles were obtained through terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA amplicons followed by sequencing. Bacterial community profiles showed that ten common terminal restriction fragments made up approximately 50% of the measured bacterial community. As much as 42% of the measured bacterial community could be monitored by using quantitative PCR and primers that targeted three dominant operational taxonomic units. Despite changes in wastewater composition and dissolved oxygen levels, the bacterial community composition appeared stable and was dominated by α-Proteobacteria and β-Proteobacteria, with a lesser amount of the highly diverse bacterial phylum Bacteroidetes. A short period of considerable change in the bacterial community composition did not appear to affect treatment performance indicating functional redundancy in this treatment system.