Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Scabies and Impetigo Prevalence and Risk Factors in Fiji: A National Survey

BackgroundScabies is recognised as a major public health problem in many countries, and is responsible for significant morbidity due to secondary bacterial infection of the skin causing impetigo, abscesses and cellulitis, that can in turn lead to serious systemic complications such as septicaemia, kidney disease and, potentially, rheumatic heart disease. Despite the apparent burden of disease in many countries, there have been few large-scale surveys of scabies prevalence or risk factors. We undertook a population-based survey in Fiji of scabies and impetigo to evaluate the magnitude of the problem and inform public health strategies.ConclusionsAs far as we are aware, this is the first national survey of scabies and impetigo ever conducted. We found that scabies occurs at high levels across all age groups, ethnicities, and geographical locations. Improved strategies are urgently needed to achieve control of scabies and its complications in endemic communities.     

N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain.

A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.     

Shrub clearing adversely affects the abundance of Ixodes ricinus ticks

In order to get a better understanding of the importance of vertical forest structure as a component of Ixodes ricinus tick habitat, an experiment was set up in a coniferous forest on sandy soils in northern Belgium. Ticks were sampled in six control and six treatment plots on various sampling occasions in 2008–2010. In the course of the study period, a moderate thinning was carried out in all plots and shrub clearing was performed in the treatment plots. Thinning had no effect on tick abundance, while shrub clearing had an adverse affect on the abundance of all three life stages (larva, nymph, adult) up to 2 years post-clearing. Our findings are especially relevant in the light of the ongoing efforts to improve vertical forest structure in Belgium and many other parts of Europe, which might create suitable habitats for ticks and change the epidemiology of tick-borne diseases. Also, our results indicate that shrub clearing could be applied as a tick control measure in recreational areas where there is a high degree of human-tick contact.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.     

Claudin 7 is reduced in uterine epithelial cells during early pregnancy in the rat

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.     

SNPflow: A Lightweight Application for the Processing,Storing and Automatic Quality Checking of Genotyping Assays

Single nucleotide polymorphisms (SNPs) play a prominent role in modern genetics. Current genotyping technologies such as Sequenom iPLEX, ABI TaqMan and KBioscience KASPar made the genotyping of huge SNP sets in large populations straightforward and allow the generation of hundreds of thousands of genotypes even in medium sized labs. While data generation is straightforward, the subsequent data conversion, storage and quality control steps are time-consuming, error-prone and require extensive bioinformatic support. In order to ease this tedious process, we developed SNPflow. SNPflow is a lightweight, intuitive and easily deployable application, which processes genotype data from Sequenom MassARRAY (iPLEX) and ABI 7900HT (TaqMan, KASPar) systems and is extendible to other genotyping methods as well. SNPflow automatically converts the raw output files to ready-to-use genotype lists, calculates all standard quality control values such as call rate, expected and real amount of replicates, minor allele frequency, absolute number of discordant replicates, discordance rate and the p-value of the HWE test, checks the plausibility of the observed genotype frequencies by comparing them to HapMap/1000-Genomes, provides a module for the processing of SNPs, which allow sex determination for DNA quality control purposes and, finally, stores all data in a relational database. SNPflow runs on all common operating systems and comes as both stand-alone version and multi-user version for laboratory-wide use. The software, a user manual, screenshots and a screencast illustrating the main features are available at http://genepi-snpflow.i-med.ac.at.