Is TSE susceptibility in sheep influenced by RPSA gene variation?

Overdraagbare spongiforme encefalopathieën (TSE’s) of prionziekten zijn een groep van dodelijke neurodegeneratieve ziektes die bij de mens en een breed scala van diersoorten voorkomen. TSE's worden veroorzaakt door besmettelijke abnormaal gevouwen eiwitten die prionen (PrPSc) worden genoemd. Wanneer een mens of dier wordt geïnfecteerd met prionen vindt een cascade van omzettingen plaats van het lichaamseigen cellulaire prioneiwit tot de abnormaal gevouwen vorm wat tot neurodegeneratieve symptomen en uiteindelijk de dood leidt. Bij schapen werd er een sterke variatie in scrapie resistentie gevonden tussen individuen die gedeeltelijk kan verklaard worden door het PRNP-genotype van het schaap. Andere kandidaatgenen kunnen echter ook een rol spelen in resistentie tegen scrapie. Bovendien kan de studie van genen die een rol spelen in de TSE pathway leiden tot de ontwikkeling van nieuwe therapieën die toegepast kunnen worden bij de mens. Een veelbelovend kandidaatgen is RPSA, een receptor die onder andere zorgt voor de binding en internalisatie van PrP in de cel. Structurele mutaties in dit gen kunnen leiden tot een vermindering van de bindingsaffiniteit tussen RPSA en PrP en kunnen daarom gevolgen hebben voor de pathogenese en verspreiding van de ziekte. Het RPSA eiwit maakt echter deel uit van een complexe gen familie die nog niet gekarakteriseerd is bij het schaap. In hoofdstuk 1 wordt een overzicht gegeven van de literatuur over TSE’s, het RPSA gen en eiwit en priontherapieën. Hoofdstuk 2 bevat de doelstellingen van dit doctoraatsonderzoek. Het algemene doel was om alle leden van de RPSA genfamilie bij schapen te identificeren en te sequeneren en vervolgens hen te karakteriseren en te beoordelen op hun functionaliteit. Van alle functionele RPSA gen familieleden wilden we nagaan of structurele variatie in hun bindingsregio met PrP invloed heeft op de bindingsaffiniteit van de receptor, wat kan leiden tot verschillen in de resistentie voor TSE’s. We identificeerden en sequeneerden 12 RPSA gen familieleden bij het schaap in hoofdstuk 3.1. De reeds beschreven gedeeltelijke genomische sequentie van het functionele RPSA gen werd bevestigd en aangevuld met de ontbrekende sequentie. Naast het functionele RPSA gen, identificeerden we 11 nieuwe leden van de schapen RPSA genfamilie. Het waren allen geproceste pseudogenen waarvan 2 semi-geprocessed en ze werden RPSAP1-RPSAP11 genoemd. De RPSA familieleden werden vergeleken met het functionele RPSA transcript. De leden verschillen van elkaar in zowel structuur als percentage sequentie identiteit met het functionele RPSA transcript. Bovendien werd het transcriptie profiel van alle RPSA familieleden bepaald door RT-PCR in 7 weefsels. Een opmerkelijk resultaat is dat 6 van de 11 pseudogenen tot transcriptie komen in tenminste één van de geteste weefsels. De karakterisatie van de RPSA familieleden toonde aan dat geen enkel ander RPSA gen familielid, naast het actieve RPSA gen, kandidaat is om een effect te hebben op de binding tussen RPSA en PrP, hetzij als gevolg van de afwezigheid van expressie, lage sequentie identiteit met RPSA of de afwezigheid van bindingsdomeinen met PrP in het tot expressie komende polypeptide. In hoofdstuk 3.2 werden naast de 12 experimenteel gevonden RPSA familieleden nog eens 37 oviene RPSA familieleden geïdentificeerd in silico. Dit brengt het totaal op 48 oviene RPSA pseudogenen. Alle nieuw ontdekte RPSA familieleden zijn geprocessed, behalve 1 semi-geprocessed pseudogen en vertonen allemaal één of meer typische kenmerken van geproceste pseudogenen. Eens de sequenties van de RPSA pseudogenen bekend waren, was het mogelijk om een mutatiestudie uit te voeren op het functionele RPSA gen zonder interferentie van pseudogenen. We voerden de mutatie analyse uit op 33 niet-verwante schapen van 7 verschillende rassen. Er werden negentien mutaties gevonden maar geen enkele structurele mutatie die een directe rol zou kunnen spelen in de RPSA-PrP interactie. Bovendien werd aangetoond dat het RPSA eiwit van schapen, geiten en runderen 100% identiek is. Omdat er geen structurele variabiliteit van het oviene RPSA eiwit werd gevonden, wilden we onderzoeken of structurele variabiliteit van het PrP eiwit invloed kan hebben op de binding tussen RPSA en PrP, wat kan leiden tot verschillen in gevoeligheid voor prionziekten tussen soorten en binnen soorten. Om de bindingssterkte tussen RPSA en PrP moleculen van verschillende soorten te beoordelen werd in hoofdstuk 4 het klonen, de eiwitsynthese in E. coli en de zuivering van PrP en RPSA recombinante eiwitten van de mens, het rund en het schaap besproken. De resultaten van deze thesis tonen aan dat de geobserveerde variatie in scrapie resistentie bij schapen niet verklaard kan worden door structurele mutaties in het RPSA gen of in andere RPSA gen familieleden en dat de geobserveerde speciesbarrière tussen herkauwers voor bepaalde prion stammen niet verklaard kan worden door structurele verschillen in de RPSA eiwitten van verschillende herkauwers

Presence of the ABCB1 (MDR1) deletion mutation causing ivermectin hypersensitivity in certain dog breeds in Belgium

Hypersensitivity to ivermectin and certain other drugs in Collies and related breeds is caused by a 4-base pair deletion mutation in the ABCB1 gene, better known as the MDR1 gene, encoding P-glycoprotein. There is no information available, however, regarding the presence of this mutation in dogs in Belgium. In this study, the ABCB1 genotype was assessed in 92 dogs of breeds suspected to possess the deletion mutation. The results indicated that the mutation was present in the Australian Shepherd, Collie, Shetland Sheepdog and Swiss White Shepherd, but was not detected in the Bearded Collies, Border Collies and German Shepherds of this study, which is in accordance with the findings in similar breed populations of other countries. In Belgium it is therefore important to take the ABCB1 genotype of the breeds involved into account, in order to use drugs in a safe and efficient manner and to improve the selection procedure in dog breeding

Characterization of the ovine ribosomal protein SA gene and its pseudogenes

Background: The ribosomal protein SA (RPSA), previously named 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR) is a multifunctional protein that plays a role in a number of pathological processes, such as cancer and prion diseases. In all investigated species, RPSA is a member of a multicopy gene family consisting of one full length functional gene and several pseudogenes. Therefore, for studies on RPSA related pathways/pathologies, it is important to characterize the whole family and to address the possible function of the other RPSA family members. The present work aims at deciphering the RPSA family in sheep. Results: In addition to the full length functional ovine RPSA gene, 11 other members of this multicopy gene family, all processed pseudogenes, were identified. Comparison between the RPSA transcript and these pseudogenes shows a large variety in sequence identities ranging from 99% to 74%. Only one of the 11 pseudogenes, i.e. RPSAP7, shares the same open reading frame (ORF) of 295 amino acids with the RPSA gene, differing in only one amino acid. All members of the RPSA family were annotated by comparative mapping and fluorescence in situ hybridization (FISH) localization. Transcription was investigated in the cerebrum, cerebellum, spleen, muscle, lymph node, duodenum and blood, and transcripts were detected for 6 of the 11 pseudogenes in some of these tissues. Conclusions: In the present work we have characterized the ovine RPSA family. Our results have revealed the existence of 11 ovine RPSA pseudogenes and provide new data on their structure and sequence. Such information will facilitate molecular studies of the functional RPSA gene taking into account the existence of these pseudogenes in the design of experiments. It remains to be investigated if the transcribed members are functional as regulatory non-coding RNA or as functional proteins

Exploratory Survey on European Consumer and Stakeholder Attitudes towards Alternatives for Surgical Castration of Piglets

Simple SummaryIn many countries, surgical castration of piglets without pain relief or anaesthesia is still common practice. Castration is performed to minimise the incidence of boar taint, a bad taste (urine/fecal like), typically present in the meat of 5 to 10% of uncastrated male pigs. It also helps to avoid aggressive and sexual behaviour. For animal welfare reasons, alternatives are being considered, and in some countries, an alternative is already practiced. One option is to perform surgical castration with anaesthesia and relieve pain. A second option is to produce male pigs without castration, which requires detection of tainted carcasses in the slaughter house. A third option is to apply immunocastration: by a two-fold injection of a vaccine, the testes function is inhibited, which reduces boar-like behaviour and avoids boar taint. In this study, we evaluated the acceptability of each of these methods in 16 countries in Europe. Of the 4 presented options, the practice of surgical castration was least accepted (32%), whilst there was a high acceptance of castration with anaesthesia (85%), followed by immunocastration (71%) and production of boars (49%). The developed questionnaire and infographic can be used in future studies to further gain insights in consumer and stakeholder attitudes on this topic.Surgical castration of piglets without pain relief is still common practice in many countries. Possible alternatives for surgical castration are application of pain relief or anaesthesia or production of boars (entire males) and immunocastrates. Each of these alternatives faces advantages and disadvantages which may result in different citizen attitudes and consumers acceptability. Understanding which practice is acceptable to whom and why may further stimulate implementation. Consumer (n = 3251) and stakeholder (n = 1027) attitudes towards surgical castration without pain relief, surgical castration with anaesthesia, immunocastration, and production of boars were surveyed from April to June 2020 via an online questionnaire in 16 countries (>175 respondents per country). Surgical castration without pain relief was separated from each of the alternatives due to animal welfare and showed the lowest acceptability (32%). Within the alternatives, a further partitioning between the alternatives was based on perceived quality and food safety, with an acceptance of 85% for applying anaesthesia, 71% for immunocastration, and 49% for boar production. Differences depending on professional involvement and familiarity with agriculture could be observed, mainly for the acceptance of surgical castration without anaesthesia, immunocastration, and boars. Castration with anaesthesia was highly accepted by all types of respondents

Experimental protocol for sea level projections from ISMIP6 stand-alone ice sheet models

Projection of the contribution of ice sheets to sea level change as part of the Coupled Model Intercomparison Project Phase 6 (CMIP6) takes the form of simulations from coupled ice sheet–climate models and stand-alone ice sheet models, overseen by the Ice Sheet Model Intercomparison Project for CMIP6 (ISMIP6). This paper describes the experimental setup for process-based sea level change projections to be performed with stand-alone Greenland and Antarctic ice sheet models in the context of ISMIP6. The ISMIP6 protocol relies on a suite of polar atmospheric and oceanic CMIP-based forcing for ice sheet models, in order to explore the uncertainty in projected sea level change due to future emissions scenarios, CMIP models, ice sheet models, and parameterizations for ice–ocean interactions. We describe here the approach taken for defining the suite of ISMIP6 stand-alone ice sheet simulations, document the experimental framework and implementation, and present an overview of the ISMIP6 forcing to be used by participating ice sheet modeling groups

The future sea-level contribution of the Greenland ice sheet: A multi-model ensemble study of ISMIP6

The Greenland ice sheet is one of the largest contributors to global mean sea-level rise today and is expected to continue to lose mass as the Arctic continues to warm. The two predominant mass loss mechanisms are increased surface meltwater run-off and mass loss associated with the retreat of marine-terminating outlet glaciers. In this paper we use a large ensemble of Greenland ice sheet models forced by output from a representative subset of the Coupled Model Intercomparison Project (CMIP5) global climate models to project ice sheet changes and sea-level rise contributions over the 21st century. The simulations are part of the Ice Sheet Model Intercomparison Project for CMIP6 (ISMIP6).We estimate the sea-level contribution together with uncertainties due to future climate forcing, ice sheet model formulations and ocean forcing for the two greenhouse gas concentration scenarios RCP8.5 and RCP2.6. The results indicate that the Greenland ice sheet will continue to lose mass in both scenarios until 2100, with contributions of 90-50 and 32-17mm to sea-level rise for RCP8.5 and RCP2.6, respectively. The largest mass loss is expected from the south-west of Greenland, which is governed by surface mass balance changes, continuing what is already observed today. Because the contributions are calculated against an unforced control experiment, these numbers do not include any committed mass loss, i.e. mass loss that would occur over the coming century if the climate forcing remained constant. Under RCP8.5 forcing, ice sheet model uncertainty explains an ensemble spread of 40 mm, while climate model uncertainty and ocean forcing uncertainty account for a spread of 36 and 19 mm, respectively. Apart from those formally derived uncertainty ranges, the largest gap in our knowledge is about the physical understanding and implementation of the calving process, i.e. the interaction of the ice sheet with the ocean. © Author(s) 2020

The future sea-level contribution of the Greenland ice sheet: a multi-model ensemble study of ISMIP6

The Greenland ice sheet is one of the largest contributors to global mean sea-level rise today and is expected to continue to lose mass as the Arctic continues to warm. The two predominant mass loss mechanisms are increased surface meltwater run-off and mass loss associated with the retreat of marine-terminating outlet glaciers. In this paper we use a large ensemble of Greenland ice sheet models forced by output from a representative subset of the Coupled Model Intercomparison Project (CMIP5) global climate models to project ice sheet changes and sea-level rise contributions over the 21st century. The simulations are part of the Ice Sheet Model Intercomparison Project for CMIP6 (ISMIP6). We estimate the sea-level contribution together with uncertainties due to future climate forcing, ice sheet model formulations and ocean forcing for the two greenhouse gas concentration scenarios RCP8.5 and RCP2.6. The results indicate that the Greenland ice sheet will continue to lose mass in both scenarios until 2100, with contributions of 90±50 and 32±17 mm to sea-level rise for RCP8.5 and RCP2.6, respectively. The largest mass loss is expected from the south-west of Greenland, which is governed by surface mass balance changes, continuing what is already observed today. Because the contributions are calculated against an unforced control experiment, these numbers do not include any committed mass loss, i.e. mass loss that would occur over the coming century if the climate forcing remained constant. Under RCP8.5 forcing, ice sheet model uncertainty explains an ensemble spread of 40 mm, while climate model uncertainty and ocean forcing uncertainty account for a spread of 36 and 19 mm, respectively. Apart from those formally derived uncertainty ranges, the largest gap in our knowledge is about the physical understanding and implementation of the calving process, i.e. the interaction of the ice sheet with the ocean

Ribosomal protein SA and its pseudogenes in ruminants : an extremely conserved gene family

The ribosomal protein SA (RPSA), also known as 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR), has been identified as a multifunctional protein, playing an important role in multiple pathologies like cancer and prion diseases. Since RPSA is involved in the binding and internalization of the prion protein, mutations in the ovine RPSA gene, influencing the RPSA-PrPC/PrPSc binding, can potentially play a part in the resistance to prion diseases. Our goal was to further characterize the complex RPSA gene family and to detect structural mutations which can play a role in this disease. In a prior study, 11 ovine pseudogenes were detected experimentally. As the whole genome shotgun ovine genome became accessible, an in silico genome-wide screening was performed and 37 new pseudogenes (36 processed and one semi-processed pseudogene) were detected, bringing the total to 48 ovine RPSA pseudogenes. Additionally, the complete bovine genome was screened in silico and 56 pseudogenes were identified. Once these sequences were known, it was possible to analyze the presence of mutations in the coding sequence and exon-flanking regions of the ovine functional full-length RPSA gene without the interference of pseudogenic sequences. Nineteen mutations were found: one in the 5' UTR, a silent one in the coding region, and seventeen in the exon-flanking regions, including an interesting mutation in the SNORA62 gene, localized in intron 4 of RPSA, leading to potential ribosomal defects. Structural mutations of the RPSA gene can be ruled out to play a role in transmissible spongiform encephalopathies but regulatory mutations still can have an effect on these diseases