MicroRNA abundance is altered in synaptoneurosomes during prion disease.

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells.

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.

The first reported case of rabbit hemorrhagic disease in Canada.

In March 2011, rabbit hemorrhagic disease (RHD) was suspected in a 1-year-old male neutered lop-eared rabbit that had acute onset liver failure. Gross pathology, histopathology, immunohistochemistry, partial nucleic acid sequencing and phylogenetic analysis of the major capsid protein (VP60) and animal inoculation studies all supported this diagnosis making it the first confirmed case of RHD in Canada.In March 2011, rabbit hemorrhagic disease (RHD) was suspected in a 1-year-old male neutered lop-eared rabbit that had acute onset liver failure. Gross pathology, histopathology, immunohistochemistry, partial nucleic acid sequencing and phylogenetic analysis of the major capsid protein (VP60) and animal inoculation studies all supported this diagnosis making it the first confirmed case of RHD in Canada.

RésuméLe premier cas signalé de maladie hémorragique du lapin au Canada. En mars 2011, la maladie hémorragique du lapin (MHL) a été suspectée chez un lapin bélier mâle castré âgé de 1 an qui a présenté l'apparition soudaine d'une insuffisance hépatique. La pathologie macroscopique, l'histopathologie, l'immunohistochimie, le séquençage partiel de l'acide nucléique et l'analyse phylogénétique de la principale protéine de la capside (VP60) et des études d'inoculation animale ont confirmé d'emblée ce diagnostic, ce qui en fait le premier cas confirmé de MHL au Canada.(Traduit par Isabelle Vallières).

A comparison of thyroxine- and polyamine-mediated enhancement of rat facial nerve regeneration.

Thyroid hormones and spermidine, a motor neuron trophic polyamine (PA), have been shown to enhance peripheral motor nerve regeneration; however, the mechanism by which these treatment modalities exert their effect is unknown. Similarities in treatment outcome suggest that these molecules may be working via a common mechanism. Such an explanation is plausible since thyroid hormone is a potent inducer of ornithine decarboxylase (ODC), which is the rate-limiting enzyme involved in polyamine synthesis. This study was designed to morphologically evaluate the effects of exogenous thyroxine and spermidine on the regeneration of the rat facial nerve. Myelinated fiber density, axonal size, and degree of myelination were assayed by light and electron microscopy 21 days following facial nerve crush. Strikingly, the two treatment modalities had identical effects on all parameters tested. Each significantly enhanced the density of myelinated axons in regenerating nerves relative to the vehicle control. In addition, relative to the control treatment, both thyroxine and spermidine significantly increased the cross-sectional area of regenerating axons (P < 0.05). Interestingly, neither of the drug treatments had any effect on remyelination at the position where this parameter was analyzed. The concurrent administration of both thyroxine and spermidine did not synergistically enhance motor neuron regeneration. These data support the hypothesis that thyroxine and spermidine enhance neural regeneration by a common mechanism.

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells.

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.