SNaPaer: A Practical Single Nucleotide Polymorphism Multiplex Assay for Genotyping of Pseudomonas aeruginosa

Multilocus sequence typing (MLST) represents the gold standard genotyping method in studies concerning microbial population structure, being particularly helpful in the detection of clonal relatedness. However, its applicability on large-scale genotyping is limited due to the high cost and time spent on the task. The selection of the most informative nucleotide positions simplifies genomic characterization of bacteria. A simple and informative multiplex, SNaPaer assay, was developed and genotyping of Pseudomonas aeruginosa was obtained after a single reaction of multiplex PCR amplification and mini-sequencing. This cost-effective technique allowed the analysis of a Portuguese set of isolates (n = 111) collected from three distinct hospitals and the genotyping data could be obtained in less than six hours. Point mutations were shown to be the most frequent event responsible for diversification of the Portuguese population sample. The Portuguese isolates corroborated the epidemic hypothesis for P. aeruginosa population. SNaPaer genotyping assay provided a discriminatory power of 0.9993 for P. aeruginosa, by testing in silico several hundreds of MLST profiles available online. The newly proposed assay targets less than 0.01% of the total MLST length and guarantees reproducibility, unambiguous analysis and the possibility of comparing and transferring data between different laboratories. The plasticity of the method still supports the addition of extra molecular markers targeting specific purposes/populations. SNaPaer can be of great value to clinical laboratories by facilitating routine genotyping of P. aeruginosa.     

Uptake of iodide in the marine haptophyte Isochrysis sp. (T.ISO) driven by iodide oxidation

Uptake of iodide was studied in the marine microalga Isochrysis sp. (isol. Haines, T.ISO) during short‐term incubations with radioactive iodide (¹²⁵I^?). Typical inhibitors of the sodium/iodide symporter (NIS) did not inhibit iodide uptake, suggesting that iodide is not taken up through this transport protein, as is the case in most vertebrate animals. Oxidation of iodide was found to be an essential step for its uptake by T.ISO and it seemed likely that hypoiodous acid (HOI) was the form of iodine taken up. Uptake of iodide was inhibited by the addition of thiourea and of other reducing agents, like L‐ascorbic acid, L‐glutathione and L‐cysteine and increased after the addition of oxidized forms of the transition metals Fe and Mn. The simultaneous addition of both hydrogen peroxide (H₂O₂) and a known iodide‐oxidizing myeloperoxidase (MPO) significantly increased iodine uptake, but the addition of H₂O₂ or MPO separately, had no effect on uptake. This confirms the observation that iodide is oxidized prior to uptake, but it puts into doubt the involvement of H₂O₂ excretion and membrane‐bound or extracellular haloperoxidase activity of T.ISO. The increase of iodide uptake by T.ISO upon Fe(III) addition suggests the nonenzymatic oxidation of iodide by Fe(III) in a redox reaction and subsequent influx of HOI. This is the first report on the mechanism of iodide uptake in a marine microalga.     

The Long Term Impact of Cataract Surgery on Quality of Life,Activities and Poverty: Results from a Six Year Longitudinal Study in Bangladesh and the Philippines

Background

Cataract surgery has been shown to improve quality of life and household economy in the short term. However, it is unclear whether these benefits are sustained over time. This study aims to assess the six year impact of cataract surgery on health related quality of life (HRQoL), daily activities and economic poverty in Bangladesh and The Philippines.

Methods and Findings

This was a longitudinal study. At baseline people aged ≥50 years with visual impairment due to cataract (‘cases’) and age-, sex-matched controls without visual impairment were interviewed about vision specific and generic HRQoL, daily activities and economic indicators (household per capita expenditure, assets and self-rated wealth). Cases were offered free or subsidised cataract surgery. Cases and controls were re-interviewed approximately one and six years later. At baseline across the two countries there were 455 cases and 443 controls. Fifty percent of cases attended for surgery. Response rates at six years were 47% for operated cases and 53% for controls. At baseline cases had poorer health and vision related QoL, were less likely to undertake productive activities, more likely to receive assistance with activities and were poorer compared to controls (p<0.05). One year after surgery there were significant increases in HRQoL, participation and time spent in productive activities and per capita expenditure and reduction in assistance with activities so that the operated cases were similar to controls. These increases were still evident after six years with the exception that time spent on productive activities decreased among both cases and controls.

Conclusion

Cataract causing visual loss is associated with reduced HRQoL and economic poverty among older adults in low-income countries. Cataract surgery improves the HRQoL of the individual and economy of the household. The findings of this study suggest these benefits are sustained in the long term.     

The Association between HMGA1 rs146052672 Variant and Type 2 Diabetes: A Transethnic Meta-Analysis

The high-mobility group A1 (HMGA1) gene has been previously identified as a potential novel candidate gene for susceptibility to insulin resistance and type 2 diabetes (T2D) mellitus. For this reason, several studies have been conducted in recent years examining the association of the HMGA1 gene variant rs146052672 (also designated IVS5-13insC) with T2D. Because of non-univocal data and non-overlapping results among laboratories, we conducted the current meta-analysis with the aim to yield a more precise and reliable conclusion for this association. Using predetermined inclusion criteria, MEDLINE, PubMed, Web of Science, Scopus, Google Scholar and Embase were searched for all relevant available literature published until November 2014. Two of the authors independently evaluated the quality of the included studies and extracted the data. Values from the single studies were combined to determine the meta-analysis pooled estimates. Heterogeneity and publication bias were also examined. Among the articles reviewed, five studies (for a total of 13,789 cases and 13,460 controls) met the predetermined criteria for inclusion in this meta-analysis. The combined adjusted odds ratio estimates revealed that the rs146052672 variant genotype had an overall statistically significant effect on increasing the risk of development of T2D. As most of the study subjects were Caucasian, further studies are needed to establish whether the association of this variant with an increased risk of T2D is generalizable to other populations. Also, in the light of this result, it would appear to be highly desirable that further in-depth investigations should be undertaken to elucidate the biological significance of the HMGA1 rs146052672 variant.     

The plant communities of theAsplenietea trichomanis in the SW Iberian Peninsula

The rupicolous vegetation of theAsplenietea trichomanis in the south-western Iberian Peninsula is analysed using the methods of the Zürich-Montpellier School. Phytosociological tables, and biogeographical, ecological and floristic data are given for a number of syntaxa. A phytosociological synopsis of the studied vegetation was made for the entire region. Four new syntaxa are described: theCoincyo logirostri-Dianthetum lusitani, Jasiono marianae-Dianthetum lusitani jasionetosum tomentosae, Digitali thapsi-Dianthetum lusitani conopodietosum ramosi andSedo hirsuti-Polypodietum cambrici polypodietosum interjecti.     

Generation of lycopene-overproducing strains of the fungus <Emphasis Type="Italic">Mucor circinelloides</Emphasis> reveals important aspects of lycopene formation and accumulation

Objectives

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.

Results

Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.

Conclusions

A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

     

p38 alpha MAPK inhibits JNK activation and collaborates with IkappaB kinase 2 to prevent endotoxin-induced liver failure

Activation of c-Jun amino-terminal kinase (JNK) facilitates tumour necrosis factor (TNF)-induced cell death. The p38 mitogen-activated protein kinase pathway is induced by TNF stimulation, but it has not been implicated in TNF-induced cell death. Here, we show that hepatocyte-specific ablation of p38alpha in mice results in excessive activation of JNK in the liver after in vivo challenge with bacterial lipopolysaccharide (LPS). Despite increased JNK activity, p38alpha-deficient hepatocytes were not sensitive to LPS/TNF toxicity showing that JNK activation was not sufficient to mediate TNF-induced liver damage. By contrast, LPS injection caused liver failure in mice lacking both p38alpha and IkappaB kinase 2 (IKK2) in hepatocytes. Therefore, when combined with partial nuclear factor-kappaB inhibition, p38alpha deficiency sensitizes the liver to cytokine-induced damage. Collectively, these results reveal a new function of p38alpha in collaborating with IKK2 to protect the liver from LPS/TNF-induced failure by controlling JNK activation.     

Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

Background  

Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required.