Involvement of a phospholipase C in the hemolytic activity of a clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Background  

Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.     

Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Hypoxic (low oxygen) and reperfusion (post‐hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent‐free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro‐octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non‐reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO‐solubilized occludin revealed that occludin oligomeric assemblies co‐localizing with ‘TJ‐associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO‐solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non‐covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide‐bonded inner core, and dispersal of these non‐covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.     

Colonisation and mass rearing: learning from others

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations.However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued - and sometimes achieved - by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs - and characteristics of which are determined - in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.     

The oxidized thiol proteome in aging and cataractous mouse and human lens revealed by ICAT labeling

Age‐related cataractogenesis is associated with disulfide‐linked high molecular weight (HMW) crystallin aggregates. We recently found that the lens crystallin disulfidome was evolutionarily conserved in human and glutathione‐depleted mouse (LEGSKO) cataracts and that it could be mimicked by oxidation in vitro (Mol. Cell Proteomics, 14, 3211‐23 (2015)). To obtain a comprehensive blueprint of the oxidized key regulatory and cytoskeletal proteins underlying cataractogenesis, we have now used the same approach to determine, in the same specimens, all the disulfide‐forming noncrystallin proteins identified by ICAT proteomics. Seventy‐four, 50, and 54 disulfide‐forming proteins were identified in the human and mouse cataracts and the in vitro oxidation model, respectively, of which 17 were common to all three groups. Enzymes with oxidized cysteine at critical sites include GAPDH (hGAPDH, Cys247), glutathione synthase (hGSS, Cys294), aldehyde dehydrogenase (hALDH1A1, Cys126 and Cys186), sorbitol dehydrogenase (hSORD, Cys140, Cys165, and Cys179), and PARK7 (hPARK7, Cys46 and Cys53). Extensive oxidation was also present in lens‐specific intermediate filament proteins, such as BFSP1 and BFSP12 (hBFSP1 and hBFSP12, Cys167, Cys65, and Cys326), vimentin (mVim, Cys328), and cytokeratins, as well as microfilament and microtubule filament proteins, such as tubulin and actins. While the biological impact of these modifications for lens physiology remains to be determined, many of these oxidation sites have already been associated with either impaired metabolism or cytoskeletal architecture, strongly suggesting that they have a pathogenic role in cataractogenesis. By extrapolation, these findings may be of broader significance for age‐ and disease‐related dysfunctions associated with oxidant stress.     

QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi?×?Leghorn F2 cross,using a medium-density SNP panel

Background

Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F₂ cross. The 860 F₂ animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied.

Results

Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL.

Conclusions

The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between different models and marker densities.     

Detection of QTL controlling digestive efficiency and anatomy of the digestive tract in chicken fed a wheat-based diet

Background

Improving digestive efficiency is a major goal in poultry production, to reduce production costs, make possible the use of alternative feedstuffs and decrease the volume of manure produced. Since measuring digestive efficiency is difficult, identifying molecular markers associated with genes controlling this trait would be a valuable tool for selection. Detection of QTL (quantitative trait loci) was undertaken on 820 meat-type chickens in a F2 cross between D- and D+ lines divergently selected on low or high AMEn (apparent metabolizable energy value of diet corrected to 0 nitrogen balance) measured at three weeks in animals fed a low-quality diet. Birds were measured for 13 traits characterizing digestive efficiency (AMEn, coefficients of digestive utilization of starch, lipids, proteins and dry matter (CDUS, CDUL, CDUP, CDUDM)), anatomy of the digestive tract (relative weights of the proventriculus, gizzard and intestine and proventriculus plus gizzard (RPW, RGW, RIW, RPGW), relative length and density of the intestine (RIL, ID), ratio of proventriculus and gizzard to intestine weight (PG/I); and body weight at 23 days of age. Animals were genotyped for 6000 SNPs (single nucleotide polymorphisms) distributed on 28 autosomes, the Z chromosome and one unassigned linkage group.

Results

Nine QTL for digestive efficiency traits, 11 QTL for anatomy-related traits and two QTL for body weight at 23 days of age were detected. On chromosome 20, two significant QTL at the genome level co-localized for CDUS and CDUDM, i.e. two traits that are highly correlated genetically. Moreover, on chromosome 16, chromosome-wide QTL for AMEn, CDUS, CDUDM and CDUP, on chromosomes 23 and 26, chromosome-wide QTL for CDUS, on chromosomes 16 and 26, co-localized QTL for digestive efficiency and the ratio of intestine length to body weight and on chromosome 27 a chromosome-wide QTL for CDUDM were identified.

Conclusions

This study identified several regions of the chicken genome involved in the control of digestive efficiency. Further studies are necessary to identify the underlying genes and to validate these in commercial populations and breeding environments.     

Incidence of Otitis Media in a Contemporary Danish National Birth Cohort

Objectives

In recent years welfare in Denmark has increased which might be expected to reduce otitis media (OM) incidence. We examined the age-specific incidence of OM in a nation-wide cohort of children aged 0–7 years born in 1996–2003 (Danish National Birth Cohort, DNBC). Only selection was ability to understand and speak Danish.

Methods

Information of OM and ventilation tubes (VT) was collected through three maternal interviews at 6-month, 18-month and 7-years of age and based on this age-specific and cumulative incidence of OM was calculated. As different numbers of the total population answered the different interviews, the calculations are done with different denominators. The information in DNBC was validated against two population based registries containing information of VT insertions.

Results

Cumulative incidence of OM at 7 years was 60.6% (31,982/52,755). For children with OM, 16.2% (7143/44194) had their first OM episodes between 0–6 months of age, 44.3% (19579/44194) between 7–18 months, and 39.5% (17472/44194) between 19 months and 7 years. Four or more OM episodes before 7 years were reported by 39.5% (12620/31982) and by 64.0% (2482/3881) of those who had their OM debut between 0–6 months; by 48.2% (4998/10378) with debut between 7–18 months; and by 28.7% (4996/17344) with debut between 19 months and 7 years. These figures are essentially unchanged from earlier figures from Denmark. VT insertion at least once was reported by 26,1% in the 7-year interview. Assuming recordings in the Danish National Patient Registry to be gold standard, maternal self-reportings in DNBC of insertion of VT showed high sensitivity (96.4%), specificity (98.2%), and positive (94.8%) and negative predictive values (98.8%).

Conclusion

OM affects nearly 2/3 of preschool children in Denmark despite reduction in known OM risk factors.     

AB5 toxins: structures and inhibitor design

High-resolution crystal structures of AB₅ toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.