Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrPSc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrPSc propagation involves conversion from its normal isoform, PrPC, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrPSen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10-8 and 10-13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrPC. Although the brains of 263K-affected mice had no immunoblot-detectable PrPRes, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrPRes strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc

Women's job quality across family life stages: An analysis of female employees across 27 European countries

There is little empirical evidence on how working conditions affect women’s employment and fertility choices, despite a number of studies on the impact of individual-level and institutional factors. The article addresses this gap by examining how family life stages are related to particular aspects of job quality among employed women in 27 European countries. The central argument of the analysis is that high-quality jobs are conducive to both transitions to motherhood and employment after childbirth as women select into these roles. Accordingly, mothers of young children, if employed, are expected to have relatively better quality jobs. Four dimensions of job quality are considered: job security, career progression, working time and intrinsic job quality. The results indicate that mothers with young children are more likely to hold high-quality jobs than women at other life stages with respect to working time quality and job security, but with some variation across countries for job security. The findings highlight the importance of high-quality jobs for women’s fertility decisions and labour market attachment after childbirth, with implications for European employment policy

Prion protein-specific antibodies that detect multiple TSE agents with high sensitivity

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays

The Importance of Craniofacial Sutures in Biomechanical Finite Element Models of the Domestic Pig

Craniofacial sutures are a ubiquitous feature of the vertebrate skull. Previous experimental work has shown that bone strain magnitudes and orientations often vary when moving from one bone to another, across a craniofacial suture. This has led to the hypothesis that craniofacial sutures act to modify the strain environment of the skull, possibly as a mode of dissipating high stresses generated during feeding or impact. This study tests the hypothesis that the introduction of craniofacial sutures into finite element (FE) models of a modern domestic pig skull would improve model accuracy compared to a model without sutures. This allowed the mechanical effects of sutures to be assessed in isolation from other confounding variables. These models were also validated against strain gauge data collected from the same specimen ex vivo. The experimental strain data showed notable strain differences between adjacent bones, but this effect was generally not observed in either model. It was found that the inclusion of sutures in finite element models affected strain magnitudes, ratios, orientations and contour patterns, yet contrary to expectations, this did not improve the fit of the model to the experimental data, but resulted in a model that was less accurate. It is demonstrated that the presence or absence of sutures alone is not responsible for the inaccuracies in model strain, and is suggested that variations in local bone material properties, which were not accounted for by the FE models, could instead be responsible for the pattern of results

Effective alcohol policies and lifetime abstinence: An analysis of the International Alcohol Control policy index.

INTRODUCTION: Alcohol abstinence remains common among adults globally, although low and middle-income countries are experiencing declines in abstention. The effect of alcohol policies on lifetime abstinence is poorly understood. The International Alcohol Control (IAC) policy index was developed to benchmark and monitor the uptake of effective alcohol policies and has shown strong associations with alcohol per capita consumption and drinking patterns. Uniquely, the index incorporates both policy 'stringency' and 'impact', reflecting policy implementation and enforcement, across effective policies. Here we assessed the association of the IAC policy index with lifetime abstinence in a diverse sample of jurisdictions. METHODS: We conducted a cross-sectional analysis of the relationship between the IAC policy index score, and its components, and lifetime abstinence among adults (15+ years) in 13 high and middle-income jurisdictions. We examined the correlations for each component of the index and stringency and impact separately. RESULTS: Overall, the total IAC policy index scores were positively correlated with lifetime abstinence (r = 0.76), as were both the stringency (r = 0.62) and impact (r = 0.82) scores. Marketing restrictions showed higher correlations with lifetime abstinence than other policy domains (r = 0.80), including restrictions on physical availability, pricing policies and drink-driving prevention. DISCUSSION AND CONCLUSION: Our findings suggest that restricting alcohol marketing could be an important policy for the protection of alcohol abstention. The IAC policy index may be a useful tool to benchmark the performance of alcohol policy in supporting alcohol abstention in high and middle-income countries

Effective alcohol policies and lifetime abstinence: An analysis of the International Alcohol Control policy index

© 2022 The Authors.Introduction Alcohol abstinence remains common among adults globally, although low and middle-income countries are experiencing declines in abstention. The effect of alcohol policies on lifetime abstinence is poorly understood. The International Alcohol Control (IAC) policy index was developed to benchmark and monitor the uptake of effective alcohol policies and has shown strong associations with alcohol per capita consumption and drinking patterns. Uniquely, the index incorporates both policy ‘stringency’ and ‘impact’, reflecting policy implementation and enforcement, across effective policies. Here we assessed the association of the IAC policy index with lifetime abstinence in a diverse sample of jurisdictions. Methods We conducted a cross-sectional analysis of the relationship between the IAC policy index score, and its components, and lifetime abstinence among adults (15+ years) in 13 high and middle-income jurisdictions. We examined the correlations for each component of the index and stringency and impact separately. Results Overall, the total IAC policy index scores were positively correlated with lifetime abstinence (r = 0.76), as were both the stringency (r = 0.62) and impact (r = 0.82) scores. Marketing restrictions showed higher correlations with lifetime abstinence than other policy domains (r = 0.80), including restrictions on physical availability, pricing policies and drink-driving prevention. Discussion and Conclusion Our findings suggest that restricting alcohol marketing could be an important policy for the protection of alcohol abstention. The IAC policy index may be a useful tool to benchmark the performance of alcohol policy in supporting alcohol abstention in high and middle-income countries

Benchmarking alcohol policy based on stringency and impact: The International Alcohol Control (IAC) policy index.

This study developed a measurement tool to assess stringency and 'on-the-ground' impact of four key alcohol policy domains to create an alcohol policy index suitable for benchmarking alcohol policy and assessing change over time in middle- and high-income countries. It involved a collaboration between researchers in 12 diverse countries: New Zealand; Australia; England; Scotland; Netherlands; Vietnam; Thailand; South Africa; Turkey; Chile; Saint Kitts and Nevis and Mongolia. Data on the four most effective alcohol policy domains (availability, pricing policy, alcohol marketing, drink driving) were used to create an alcohol policy index based on their association with alcohol per capita consumption (APC) of commercial (recorded) alcohol. An innovation was the inclusion of measures of impact along with the stringency of the legislation or regulation. The resulting International Alcohol Control (IAC) Policy Index showed a very high negative correlation (-0.91) with recorded APC. Greater affordability of alcohol, an impact measure taking into account prices paid and countries' Gross Domestic Product, was predictive of higher APC (-0.80). Countries in which more modes of alcohol marketing are legally allowed and used had higher APC. Legislation on outlet density and drink driving predicted APC whereas trading hours did not. While stringency and impact measures varied between domains in terms of relationship with APC, overall, there was a strong correlation between impact and stringency (0.77). The IAC Policy Index, which includes measures of policy stringency and 'on-the-ground' impacts in relation to four key policy areas, was found to be strongly associated with commercial alcohol consumed in a number of diverse country settings. It showed a larger relationship than previous indices that include more policy dimensions. The index provides a relatively simple tool for benchmarking and communication with policy makers to encourage a strong focus on uptake of these four most effective alcohol policies.fals

Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrPSc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrPSc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 103.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrPSc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants