CHCHD2 harboring Parkinson's disease-linked T61I mutation precipitates inside mitochondria and induces precipitation of wild-type CHCHD2

The T61I mutation in CHCHD2, a protein residing in the mitochondrial intermembrane space, causes an autosomal dominant form of Parkinson’s disease (PD), but the underlying pathogenic mechanisms are not well understood. Here, we compared the subcellular localization and solubility of wild-type and T61I mutant CHCHD2 in human cells. We found that mitochondrial targeting of both wild-type and T61I CHCHD2 depended on the four cysteine residues in the C-terminal coiled-coil-helix-coiled-coil-helix (CHCH) domain but not on the N-terminal predicted mitochondrial targeting sequence. The T61I mutation did not interfere with mitochondrial targeting of the mutant protein, but induced its precipitation in the IMS. Moreover, T61I CHCHD2 induced increased mitochondrial production of reactive oxygen species (ROS) and apoptosis, which was prevented by treatment with anti-oxidants. Retention of T61I CHCHD2 in the cytosol through mutation of the cysteine residues in the CHCH domain prevented its precipitation as well as its apoptosis-inducing effect. Importantly, T61I CHCHD2 potently impaired the solubility of wild-type CHCHD2. In conclusion, our data show that the T61I mutation renders mutant CHCHD2 insoluble inside mitochondria, suggesting loss of function of the mutant protein. In addition, T61I CHCHD2 exerts a dominant-negative effect on the solubility of wild-type CHCHD2, explaining the dominant inheritance of this form of PD

Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on succesive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, φKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee

Characterization of the Adhesin of Escherichia coli F18 Fimbriae

Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein

Vers un monitoring des facteurs de risque d'émergence des maladies animales?

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Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses36524

In this study, conducted at five slaughterhouses, individual pigs were sampled and followed up from stunning to cooling down of the carcasses. In this way, Salmonella prevalence and possible risk points were described. At the lairage area, pens were sampled using overshoes. At stunning and bleeding, pigs were individually identified and subsequently swabs were taken of the oral cavity and the carcass after polishing, splitting and forced chilling. Additionally, duodenum, ileum, rectum and mesenteric lymph nodes were extracted and samples were taken of the scalding water. All samples were submitted to Salmonella isolation and Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). Of all samples taken (n = 1953), 14.1% were Salmonella positive. The prevalence of S. in the lairage area varied widely (from 0 to 100%) between the slaughterhouses. Of the sampled pigs (n = 226), 48.2% were positive in at least one sample. Statistical analysis revealed that the contamination of the lairage area was related to a higher amount of positive carcasses after polishing. Furthermore, the contamination of the carcasses after splitting and forced chilling was related to the contamination level of the carcass after polishing. A relation between the outer (carcass) contamination and the inner (gut content and lymph nodes) contamination of a pig could not be established. The predominant serotypes were S. Typhimurium (58.7%) and S. Derby (17.4%). Genotyping revealed 46 different PFGE profiles among the 276 Salmonella isolates. The same genotype at the lairage area as in the oral cavity of the pigs was found in 95%. The results indicate that the lairage area is a primary source of Salmonella in slaughter pigs and that carcass contamination originates from the environment rather than from the pig (inner contamination) itself. It further shows that slaughterhouses vary in their capability of dealing with Salmonella positive pigs. A slaughterhouse specific approach is needed, however, general guidelines should be provided to decrease the contamination level of the lairage area and the slaughter environment</p

A multiplex PCR to identify porcine mycoplasmas present in broth cultures.

&lt;p&gt;Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.&lt;/p&gt;</p