Mutant PrP is delayed in its exit from the endoplasmic reticulum,but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.     

Vaccination of vampire bats using recombinant vaccinia-rabies virus

Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested.     

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G0₀ arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.     

Spatial Heterogeneity as a Genetic Mixing Mechanism in Highly Philopatric Colonial Seabirds

How genetic diversity is maintained in philopatric colonial systems remains unclear, and understanding the dynamic balance of philopatry and dispersal at all spatial scales is essential to the study of the evolution of coloniality. In the King penguin, Aptenodytes patagonicus, return rates of post-fledging chicks to their natal sub-colony are remarkably high. Empirical studies have shown that adults return year after year to their previous breeding territories within a radius of a few meters. Yet, little reliable data are available on intra- and inter-colonial dispersal in this species. Here, we present the first fine-scale study of the genetic structure in a king penguin colony in the Crozet Archipelago. Samples were collected from individual chicks and analysed at 8 microsatellite loci. Precise geolocation data of hatching sites and selective pressures associated with habitat features were recorded for all sampling locations. We found that despite strong natal and breeding site fidelity, king penguins retain a high degree of panmixia and genetic diversity. Yet, genetic structure appears markedly heterogeneous across the colony, with higher-than-expected inbreeding levels, and local inbreeding and relatedness hotspots that overlap predicted higher-quality nesting locations. This points towards heterogeneous population structure at the sub-colony level, in which fine-scale environmental features drive local philopatric behaviour, while lower-quality patches may act as genetic mixing mechanisms at the colony level. These findings show how a lack of global genetic structuring can emerge from small-scale heterogeneity in ecological parameters, as opposed to the classical model of homogeneous dispersal. Our results also emphasize the importance of sampling design for estimation of population parameters in colonial seabirds, as at high spatial resolution, basic genetic features are shown to be location-dependent. Finally, this study stresses the importance of understanding intra-colonial dispersal and genetic mixing mechanisms in order to better estimate species-wide gene flows and population dynamics.     

Adaptive Potential of Hybridization among Malaria Vectors: Introgression at the Immune Locus TEP1 between Anopheles coluzzii and A. gambiae in ‘Far-West’ Africa

“Far-West” Africa is known to be a secondary contact zone between the two major malaria vectors Anopheles coluzzii and A. gambiae. We investigated gene-flow and potentially adaptive introgression between these species along a west-to-east transect in Guinea Bissau, the putative core of this hybrid zone. To evaluate the extent and direction of gene flow, we genotyped site 702 in Intron-1 of the para Voltage-Gated Sodium Channel gene, a species-diagnostic nucleotide position throughout most of A. coluzzii and A. gambiae sympatric range. We also analyzed polymorphism in the thioester-binding domain (TED) of the innate immunity-linked thioester-containing protein 1 (TEP1) to investigate whether elevated hybridization might facilitate the exchange of variants linked to adaptive immunity and Plasmodium refractoriness. Our results confirm asymmetric introgression of genetic material from A. coluzzii to A. gambiae and disruption of linkage between the centromeric "genomic islands" of inter-specific divergence. We report that A. gambiae from the Guinean hybrid zone possesses an introgressed TEP1 resistant allelic class, found exclusively in A. coluzzii elsewhere and apparently swept to fixation in West Africa (i.e. Mali and Burkina Faso). However, no detectable fixation of this allele was found in Guinea Bissau, which may suggest that ecological pressures driving segregation between the two species in larval habitats in this region may be different from those experienced in northern and more arid parts of the species’ range. Finally, our results also suggest a genetic subdivision between coastal and inland A. gambiae Guinean populations and provide clues on the importance of ecological factors in intra-specific differentiation processes.     

Electronic Cigarettes Efficacy and Safety at 12 Months: Cohort Study

Objective

To evaluate the safety and efficacy as a tool of smoking cessation of electronic cigarettes (e-cigarettes), directly comparing users of e-cigarettes only, smokers of tobacco cigarettes only, and smokers of both.

Design

Prospective cohort study. Final results are expected in 2019, but given the urgency of data to support policies on electronic smoking, we report the results of the 12-month follow-up.

Data Sources

Direct contact and structured questionnaires by phone or via internet.

Methods

Adults (30–75 years) were included if they were smokers of ≥1 tobacco cigarette/day (tobacco smokers), users of any type of e-cigarettes, inhaling ≥50 puffs weekly (e-smokers), or smokers of both tobacco and e-cigarettes (dual smokers). Carbon monoxide levels were tested in a sample of those declaring tobacco smoking abstinence.

Main Outcome Measures

Sustained smoking abstinence from tobacco smoking at 12 months, reduction in the number of tobacco cigarettes smoked daily.

Data Synthesis

We used linear and logistic regression, with region as cluster unit.

Results

Follow-up data were available for 236 e-smokers, 491 tobacco smokers, and 232 dual smokers (overall response rate 70.8%). All e-smokers were tobacco ex-smokers. At 12 months, 61.9% of the e-smokers were still abstinent from tobacco smoking; 20.6% of the tobacco smokers and 22.0% of the dual smokers achieved tobacco abstinence. Adjusting for potential confounders, tobacco smoking abstinence or cessation remained significantly more likely among e-smokers (adjusted OR 5.19; 95% CI: 3.35–8.02), whereas adding e-cigarettes to tobacco smoking did not enhance the likelihood of quitting tobacco and did not reduce tobacco cigarette consumption. E-smokers showed a minimal but significantly higher increase in self-rated health than other smokers. Non significant differences were found in self-reported serious adverse events (eleven overall).

Conclusions

Adding e-cigarettes to tobacco smoking did not facilitate smoking cessation or reduction. If e-cigarette safety will be confirmed, however, the use of e-cigarettes alone may facilitate quitters remaining so.

Registration Number

NCT01785537.     

Estimation of anisotropy coefficient of swine pancreas,liver and muscle at 1064 nm based on goniometric technique

Optical properties of tissues are required for theoretical modeling of Laser Ablation in tumor therapy. The light scattering characteristic of tissues is described by the anisotropy coefficient, g. The relationship between the angular distribution of scattered light and g is given by the Henyey‐Greenstein (HG) phase function. This work describes the estimation of anisotropy coefficients of ex vivo swine pancreas, liver and muscle at 1064 nm. The intensities of scattered light at fixed angles were measured under repeatability conditions. Experimental data were fitted with a two‐term HG, estimating the anisotropy coefficients for the forward (e.g., 0.956 for pancreas, 0.964 for liver and 0.968 for muscle) and the backward (e.g., –0.481 for pancreas, –0.414 for liver and –0.372 for muscle) scattering.

Experimental set up employed to estimate the anisotropy coefficient of biological tissues. The image on the left depicts the holder used to house tissue, laser fiber and photodetector; on the left an example of scattered light beam is shown, as well as the effect due to Snell's law.     

Cerebral Circulation Time is Prolonged and Not Correlated with EDSS in Multiple Sclerosis Patients: A Study Using Digital Subtracted Angiography

Literature has suggested that changes in brain flow circulation occur in patients with multiple sclerosis. In this study, digital subtraction angiography (DSA) was used to measure the absolute CCT value in MS patients and to correlate its value to age at disease onset and duration, and to expand disability status scale (EDSS). DSA assessment was performed on eighty MS patients and on a control group of forty-four age-matched patients. CCT in MS and control groups was calculated by analyzing the angiographic images. Lesion and brain volumes were calculated in a representative group of MS patients. Statistical correlations among CCT and disease duration, age at disease onset, lesion load, brain volumes and EDSS were considered. A significant difference between CCT in MS patients (mean = 4.9s; sd = 1.27s) and control group (mean = 2.8s; sd = 0.51s) was demonstrated. No significant statistical correlation was found between CCT and the other parameters in all MS patients. Significantly increased CCT value in MS patients suggests the presence of microvascular dysfunctions, which do not depend on clinical and MRI findings. Hemodynamic changes may not be exclusively the result of a late chronic inflammatory process.