PECAM‐1 supports leukocyte diapedesis by tension‐dependent dephosphorylation of VE‐cadherin

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE‐cadherin‐Y731. Here, we reveal the underlying mechanism. Leukocyte‐induced stimulation of PECAM‐1 triggers dissociation of the phosphatase SHP2 which then directly targets VE‐cadherin‐Y731. The binding site of PECAM‐1 for SHP2 is needed for VE‐cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM‐1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE‐cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca²⁺‐signaling, non‐muscle myosin II activation, and endothelial cell tension. Since we found that β‐catenin/plakoglobin mask VE‐cadherin‐Y731 and leukocyte docking to endothelial cells exert force on the VE‐cadherin–catenin complex, we propose that leukocytes destabilize junctions by PECAM‐1‐SHP2‐triggered dephosphorylation of VE‐cadherin‐Y731 which becomes accessible by actomyosin‐mediated mechanical force exerted on the VE‐cadherin–catenin complex.     

Late presentation of recurrent syncope after permanent pacemaker implantation due to Lead−Header malapposition

Permanent pacemaker (PPM) malfunction due to electrical connection problems such as a loose set screw or lead-header malapposition is extremely rare. We present a patient with complete heart block (CHB) who had PPM malfunction and recurrent syncope, late (14 months) after initial implantation, which was caused by the ventricular lead pin disengagement from the header resulting in oversensing due to noise, pacing inhibition and recurrent syncope. PPM due to lead-header malapposition this late after device implantation has previously not been reported.     

Performance of the predatory mite Amblydromalus limonicus on factitious foods

Amblydromalus limonicus Garman & McGregor (Acari: Phytoseiidae) is a generalist predatory mite with economic potential to control thrips and whiteflies in protected cultivation. We tested the development and reproduction of A. limonicus on three food sources with potential for use in laboratory production or to support its populations in a crop: fresh cattail pollen, Typha latifolia L. (Poales: Typhaceae), dry decapsulated cysts of the brine shrimp Artemia franciscana Kellogg (Branchiopoda: Artemiidae) and frozen eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). The diets were tested both on an artificial substrate and on kidney bean leaves. In the absence of food, all larvae died on the artificial substrate, whereas they succeeded in reaching the protonymphal stage on bean leaves. Immature survival was high (>90 %) on all diet–substrate combinations, except when E. kuehniella eggs were offered on the artificial substrate (35 % survival). Both sexes showed the fastest development when offered E. kuehniella eggs on leaf discs, followed by A. franciscana cysts, whereas the slowest development was achieved on T. latifolia pollen. Fecundity and oviposition rate were higher on E. kuehniella and A. franciscana than on T. latifolia. Amblydromalus limonicus females lived longer on the leaf discs than on the artificial substrates. The intrinsic rate of increase (r_m) was highest when E. kuehniella eggs were offered on leaf discs (0.256 females per female per day), whereas the lowest rate (0.128 females per female per day) was obtained when the eggs were provided on artificial substrates. The intrinsic rate of increase on A. franciscana cysts was not affected by substrate and averaged 0.22 females per female per day. Diet significantly influenced the size of A. limonicus females as measured by the distance between specific setae on the dorsal shield of the idiosoma. The application of the investigated food sources to sustain a colony of predatory mites upon their release in a greenhouse crop is discussed.     

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.     

Induction of Adipocyte Differentiation by Polybrominated Diphenyl Ethers (PBDEs) in 3T3-L1 Cells

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBPα, PPARγ, and LXRα were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis.     

Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.     

Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa,via Mutagenesis of Fatty Acid,Glycerol and Choline Degradation Pathways,Confirms the Importance of This Nutrient Source In Vivo

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.     

Artificial and factitious foods support the development and reproduction of the predatory mite Amblyseius swirskii

The generalist predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was reared on Ephestia kuehniella Zeller eggs (Lepidoptera: Pyralidae), decapsulated dry cysts of the brine shrimp Artemia franciscana Kellogg (Anostraca: Artemiidae), and on meridic artificial diets (composed of honey, sucrose, tryptone, yeast extract, and egg yolk) supplemented with pupal hemolymph of the Chinese oak silkworm Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) (AD1), with E. kuehniella eggs (AD2) or with A. franciscana cysts (AD3). Development, reproduction and predation capacity of the predatory mites were assessed in the first (G1) and sixth generation (G6) of rearing on the different diets. Immature survival rates in G1 were similar on all diets (96.8–100 %). After six generations, however, survival of A. swirskii was significantly reduced on all diets except on A. franciscana cysts. Oviposition rates did not differ between generations when females were fed on E. kuehniella, AD2 or AD3. The total number of deposited eggs was similar among diets except in G6 where the females fed on A. franciscana cysts produced more eggs than those maintained on E. kuehniella eggs. On most diets the intrinsic rates of increase in G1 were superior to those in G6, except for predators supplied with A. franciscana cysts where no differences were observed among generations. Female mites did not lose their capacity to kill first instar Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) after six generations on the different diets, but predation rates in G6 on E. kuehniella were lower than in G1. In conclusion, the different factitious and artificial diets tested in the present study supported the development and reproduction of A. swirskii for a single generation but fitness losses occurred to a varying degree after several generations on E. kuehniella eggs or the artificial diets. Artificial diet enriched with A. franciscana cysts yielded better results than the other artificial diets. Amblyseius swirskii performed best on decapsulated Artemia cysts indicating their potential for use in the mass production of the predator or to sustain its populations in the crop after release.