Microbially influenced corrosion of galvanized steel pipes in aerobic water systems

Aims: To investigate the role of heterotrophic bacteria in the corrosion of galvanized steel in the presence of water. Methods and Results: Samples were taken from corroding galvanized steel pipes conveying water for specialist applications, and heterotrophic bacteria were isolated and cultured. The majority of bacteria were Gram‐negative aerobes and included Pseudomonas sp., Bacillus pumilus, Afipia spp. and Blastobacter denitrificans/Bradyrhizobium japonicum. Zinc tolerance was assessed through growth and zinc disc diffusion experiments. In general, zinc negatively influenced growth rates. An unidentified yeast also isolated from the system demonstrated a high tolerance to zinc at concentrations up to 4 g l^?1. Coupon experiments were performed to assess corrosion by the bacteria on galvanized steel and steel coupons. The majority of isolates as pure culture biofilms (69%) accelerated corrosion of galvanized coupons, assessed as zinc release, relative to sterile control coupons (P < 0·05). Pure culture biofilms did not increase the corrosion of steel, with four isolates demonstrating protective effects. Conclusions: Pure culture biofilms of heterotrophic bacteria isolated from a corroding galvanized pipe system were found to accelerate the corrosion of galvanized steel coupons. Significance and Impact of the Study: Microbially influenced corrosion is a potential contributor to sporadically occurring failures in galvanized steel systems containing water. Management strategies should consider microbial control as a means for corrosion prevention in these systems.     

The effect of bovine diet on Salmonella survival in synthetic abomasal fluid

Aims: To investigate the effect of diet on the survival of Salmonella in the bovine abomasum. Methods and Results: Five fistulated cows were randomly assigned to one of five diets denoted as: (i) 100% grass, (ii) grass + 5·3 kg DM concentrate, (iii) 100% grass silage, (iv) 100% hay and (v) maize/grass silage plus concentrates. Rumen fluid was harvested from each dietary treatment and inoculated with nonacid (NA) and acid‐adapted (AA) 5‐strain Salmonella cocktails. After 24‐h incubation period, Salmonella were acid challenged to synthetic abomasum fluid (SAF, pH 2·5) for 5 h to determine their resistance to low pH. The study found that the volatile fatty acids composition and the pH profile of bovine rumen fluid were significantly altered (P < 0·05) by some of the dietary treatments but not others. Regression analysis found that significantly higher numbers of acid‐adapted Salmonella survived in SAF after incubation in rumen fluid from diets 1, 2 and 4, but fewer significant differences were found between diets for nonacid–adapted Salmonella. The results suggest that the acid‐adapted cells were subjected to a higher level of cell injury than the nonadapted cells. Conclusions: Pre‐incubation in rumen fluid did influence the resistance of nonacid and acid‐adapted Salmonella to SAF but it was dependant on the dietary treatment fed to the cows. Significance and Impact of the Study: This study examined the use of diet, as a modulating factor to limit the bovine excretion of Salmonella with a view to providing a scientific basis for the design of dietary management controls in the future.     

Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10⁻⁵) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10⁻⁵). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10⁻¹⁰, odds ratio 1.15 [1.10–1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04–1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression.     

Dual-mode phospholipid regulation of human inward rectifying potassium channels

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using ⁸⁶Rb⁺ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP₂) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP₂ increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP₂, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP₂ and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

High resolution imaging to assess oilseed species?? root hair responses to soil water stress

An imaging method was developed to evaluate crop species differences in root hair morphology using high resolution scanners, and to determine if the method could also detect root hair responses to soil water availability. High resolution (1890 picture elements (pixels) cm^?1) desktop scanners were buried in containers filled with soil to characterize root hair development under two water availability levels (?63 and ?188?kPa) for canola (Brassica napus L. cv Clearwater), camelina (Camelina sativa L. Crantz cv Cheyenne), flax (Linum usitatissimum L. cv CDC Bethune), and lentil (Lens culinaris Medik. cv Brewer). There was notable effect of available moisture on root hair geometry (RHG). At ?188?kPa, length from the root tip to the root hair initiation zone decreased and root hair length (RHL) became more variable near the root hair initiation zone as compared to ?63?kPa. For the response of primary axial RHL, significant main effects were present for both water availability (P?<?0.05) and species (P?<?0.0001); lateral RHL showed a significant main effect for both water availability (P?<?0.05) and species (P?<?0.01) as well. For both primary axial and lateral root hair density (RHD), there was a significant effect of species (P?<?0.0001), but no significant response to water availability. No water availability x species interaction was present in any case. Low available water reduced RHL in both primary axial and lateral roots. The change in RHL due to water availability was most evident in canola and camelina. Additionally, those with greater RHL $ \left( {\text{canola} = \text{camelina} > \text{flax} = \text{lentil}} \right) $ had lower RHD $ \left( {\text{canola} = \text{camelina} < \text{flax} < \text{lentil}} \right) $ in primary axial roots and a similar trend was found in lateral RHL. Both water and species had a significant effect on primary axial root surface area (RSA) (P?<?0.05) but no significant effect was found for lateral RSA. For primary axial RSA the longest and most dense root hair had the greatest RSA. This novel approach to in situ rhizosphere imaging allowed observation of species differences in root hair development in response to water availability and should be useful in future studies of rhizosphere interactions and crop water and nutrient management.     

Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.