Name: assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay
Uploaded: 2015-03-10T14:00:00.000+00:00
Duration: 11 min 41 s
Description: Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

USGS 국립 야생 동물 보건 센터에서 실시 동물 연구는 실험 동물 복지 가이드 라인의 NIH 사무실에 따른 제도적 동물 관리 및 사용위원회 프로토콜 # 1 EP080716에서 수행되었다. 수렵 채취 동물의 조직은 위스콘신 또는 천연 자원의 미시간 부서에서 선물했다. 1. 솔루션 준비 참고 : 아래에 나열된 솔루션은 아래 제 2의 CER 분석 기판의 준비가 필요합니다. 높은 농도의 원액에서 각 솔루션을 준비; 원액에 대한 권장 농도는 나열된 각각의 화학 물질 이름 뒤에 괄호 안에 표시됩니다. 사용까지 4 ° C에서 기판 준비 및 저장시 신선한 모든 솔루션을 준비합니다. <ol><li> 100 mM의 S : 용해 버퍼를 들어, 10 mM의 트리스, pH가 7.5 (1 M 트리스이, pH가 7.5 원액 권장) 다음의 용액을 조제비난의 클로라이드 (염화나트륨, 1 M 스톡 용액), 10 mM의 에틸렌 디아민 테트라 아세트산 (EDTA, 500 mM의 EDTA, pH가 8.0 스톡 용액), 0.5 % NP-40 (10 % 스톡 용액), 0.5 % 데 옥시 콜레이트 (DOC, 10 % 원액 ). </li><li> 변환 버퍼, 0.05 % 도데 실 황산나트륨 (SDS, 10 % 원액) 0.5 % 트리톤 X-100 1 인산염 완충 식염수 (10 % 스톡 용액) (PBS, 10, pH가 7.4 스톡 용액)의 용액을 제조 하였다. </li><li> 카오 솔루션의 경우, 염산 구아니딘 1X PBS에서 (GdnHCl, 8 M 주식) (10 배 원액)의 두 개의 3 M 솔루션을 준비합니다. 진한 염산 (HCL)을 사용하여 3.5 ± 0.05 솔루션 중 하나의 pH를 조정한다. 제 2 용액의 pH가 7.4 ± 0.05에 남아 있는지 확인하고 진한 염산이나 수산화 나트륨 (NaOH를) 필요로 조정합니다. </li></ol> 2. CER 분석 기판 쌍을 준비 <ol><li> 관심의 종에서 건강하고, 비 TSE에 감염된 뇌 조직을 얻고 10 %의 중량 당 볼륨을 준비다음 방법 중 하나를 사용하여 용해 버퍼에 균질 (/ V w) : 다운스, 비드 밀, 또는 박격포와 유 봉 균질화. 또한, 주사​​기 및 바늘로 균질화를 실시하고, 기판은 흡인 27 G 바늘 주사기를 통해 용이하게 배출 될 수있을 때까지 증가 게이지의 바늘을 사용하여 생성 된 균질 물을 구체화. <ol><li> 설치류 및 다른 작은 포유 동물로부터 제조 된 기판의 경우, 전체 뇌 (들)을 균질화; 큰 포유 동물로부터 제조 된 기판에 대해, 균질을 준비하는 OBEX (뇌간) 조직을 사용합니다. 뇌 파쇄 액의 양을 선택할 때, 단계 2.6에서 단백질을 침전 원심 분리기의 용량의 50 % 이하를 준비하지 않는다. </li><li> 취득 된 뇌 조직의 품질이 의심스러운 경우, SDS-PAGE 및 면역 블롯 (16)는 기판 (17)에 의해 제조하기 전에 PRP C 수준을 평가. </li></ol></li><li> 별도의 표시 플라스틱 원뿔 튜브에 두 개의 동일한 볼륨으로 뇌 균질 나눈다. (GdnHCl 추가뇌 균질 1 부피비로 pH를 3.5 또는 1 각 튜브에 7.4 중 하나에서 1 PBS에 3 M 솔루션). </li><li> 두 용액의 pH 값을 확인합니다. 진한 HCl을 사용하여 pH 0.05 ± 3.5로 pH를 기판의 pH를 조정한다. 다른 기판의 pH가 7.4 ± 0.05에 남아 있는지 확인하고 염산 또는 필요에 따라 NaOH로 필요에 따라 산도를 조정합니다. 산 또는 염기의 현명한 첨가하여 pH 값을 오버 슈트하지 마십시오. </li><li> 5 시간 동안 실온 (RT)에서 끝을 통해 엔드 믹서에 솔루션을 돌립니다. </li><li> 각 원추형 튜브 내의 기판 용액에 메탄올 4 권을 첨가하여 단​​백질을 침전, 와류 잘 혼합하고, 16 ~ 18 시간 동안 -20 ° C에서 배양. </li><li> 4 ℃에서 30 분 동안 13,000 × g에서 원심 분리하여 침전물 샘플. 조심스럽게 가만히 따르다하고 상층 액을 버리고 메탄올 펠릿에서 증발 할 수 있습니다. 튜브의 내부에 달라 붙었다 메탄올을 흡수을 지원하기 위해 면봉을 사용합니다. 펠렛을 통해-DR을 허용하지 마십시오Y와 메탄올이 더 이상 표시되면, 다음 단계로 진행합니다. </li><li> 변환 버퍼에 재현 탁 단백질 펠렛. 단계 2.2에서 분배 된 각각의 튜브 내로 뇌 균질 출발 물질의 양과 동일한 변환 버퍼의 양을 사용한다. 참고 : 변환 버퍼가 모두 pH를 3.5 및 7.4 처리 기판 준비에서 단백질 펠렛을 재현 탁하기 (세제의 낮은 수준과 생리적 산도를 포함) 위의 단계 1.2에 정의 된 솔루션이다 사용하는 것이 중요합니다. </li><li> cuphorn 초음파 처리기에서 간단히 초음파 처리 기판 솔루션 (30 % 최대 전력 ~ 10 초), 0.5로 나누어지는 - 표시 1.5 ml의 마이크로 원심 튜브에 1.0 ml의 볼륨. 각각의 기판의 분취 액에 품질 관리 면역 블롯 (단계 290)를 수행한다. CER 분석 (4 절)을 수행 할 준비가 될 때까지 -80 ° C에서 다른 분량을 저장합니다. </li><li> 사용 전에 CER 기판 쌍 (도 2)의 품질 관리를 수행한다. 이 단계는 CER 기판이 제공하기 것을 보장변환 연구에서 전자 최적의 결과. <ol><li> 두 개의 기판에 PRP C의 비교 금액을 확인합니다. 10 ~ 25 SDS-PAGE (16)에 의해 각각의 기판 ㎕의 17을 발현 수준에 PRP 수준을 비교. 7.4 및 3.5의 기판이 서로의 ~ 10 % 이내의 pH 단백질 수준은, 기판 쌍 CER 연구에 사용하기 적합한 지. 참고 : PRP 면역 블롯 광범위한 PRP C 저하의 증거를 보여주지해야한다. PRP 면역은 주로&gt; 20 kDa의해야합니다. 이 분자량보다 작은 밴드 분해 산물이 될 수 있습니다. 줄무늬 패턴은 기판 쌍 사이 거의 동일해야합니다. </li><li> 양 기판의 PRP C 민감한 PK되어야 같이, 100 ㎍ / ml의 최종 농도 PK 각 기판의 10-25 μl를 치료하고 열 셰이커로 1 시간 동안 37 ℃에서 1,000 rpm으로 샘플을 소화. SDS-PAGE (16, 17)을 발현 수준으로 남아있는 PRP 신호를 평가합니다.PRP 해상도와 기판은 CER 연구에서 사용하기에 적합하지 않다. </li></ol></li></ol> 3. TSE 에이전트 씨앗을 준비 <ol><li> 관심의 종 TSE에 감염된 뇌 조직을 확보하고 1X PBS pH를 7.4 균질 위의 단계 2.1에 나와있는 방법을 사용하여 (/ V는 w) 10 %를 준비합니다. 참고 : 씨앗은 또한 중추 신경계 외부의 다른 TSE에 감염된 조직에서 생산 될 수있다, 그러나, TSE에 감염된 말초 조직에서 파생 된 종자에 의한 뇌 유래 된 기판의 변환 효율은 아직 실험적으로 출간 된 문헌에서 평가되지 않았습니다. </li><li> (100)에 균질 (들) 결과로 나누어지는 - 전까지 -80 ° C에서 0.5 ml의 마이크로 원심 튜브에 300 μL 용량의 저장은 CER 분석을 수행 할 수 있습니다. </li></ol> 4. CER의 분석 주 : CER 분석은 하나의 종 ( &quot;시드&quot;에서 제공) PRP TSE 비교적 소량의 첨가를 포함부분적으로 pH를 3.5 또는 7.4 중 하나에서 변성 된 다른 종 (감염되지 않은 뇌 &quot;기판&quot;에서 제공) PRP C의 상대 초과로. 확장 흔들어 배양 기간 후, 각 반응에 PRP TSE에 의한 PRP C의 템플릿 기반의 변환은 PRP 입술 나머지 PK의 소화와 면역 검출 이후의 농도계 신호에 의해 평가된다. 이전에 pH를 3.5 대 7.4 변성 기판에 PRP 입술 레벨을 비교하는 종 장벽의 측정치를 제공한다. 상기 분석 과정의 실험 개요도 1에 제공된다. <ol><li> 천천히 감염되지 않은 CER 기판 쌍을 해동 얼음 침대 (약 1 시간 해동 시간)의 상단에 배치하여 및 TSE에 감염된 종자 솔루션 (분석은 이전에 모두 pH가 3.5과 7.4에서 변성 기판 같은 부피 필요). </li><li> 완전 해동 후에 대기음 후 통해 각 기질 용액을 추방27 G 주사기 바늘을 여러 번을 다시 균질화한다. 간단히 30 %, 최대한의 권력 ~ 10 초 동안 각 TSE에 감염된 종자 솔루션을 초음파 처리. 전환 반응을 준비하는 동안 얼음에 모든 솔루션을 보관하십시오. </li><li> TSE 에이전트 당 라벨 5 개인 낮은 바인딩, 얇은 PCR 튜브 테스트되고. </li><li> pH를 3.5로 제조하여 pH 7.4 및 (b) 기판에 CER 제조 (a) 기판의 CER 95 μL에 10 % TSE 감염된 시드 용액 5 μl를 첨가하여 이들 튜브 전환 반응을 준비한다. <ol><li> 각각의 실험 예를 들어, 이전에 모두 pH를 3.5 및 7.4에 변성 CER 기판 쌍의 평행 반응을 준비합니다. </li><li> 경험적인 결정을 통해 감염되지 않은 뇌 기판에 TSE 에이전트 씨의 비율을 최적화합니다. 일반 씨앗 : 고용 기판 비율은 100 μL의 총 반응 볼륨을 유지하고 1:99 μL, 2:98 μL 및 5:95 μl를 포함한다. 대부분의 응용 프로그램, 5:95 μL 씨 : 기판의 부피 비율이 적절한 지와 일에 무엇을 제시한다프로토콜입니다. </li></ol></li><li> 다음 TSE 화제 당 세 개의 대조 시료 시험되는 준비 : (a) 입력 PRP 입술 용 컨트롤로 95 μL 변환 버퍼에 10 % TSE 감염된 시드 용액 5 μL를 추가; (b) pH가 3.5 이상, 불특정 템플릿 변환 컨트롤 등의 (c) pH를 7.4로 제조 95 μL 기판 5 μL 변환 버퍼를 추가한다. </li><li> 간단히 잘 씨와 기판을 혼합 폐쇄 PCR 튜브에 각각의 샘플을 소용돌이. PCR 튜브의 뚜껑에 갇혀 반응 혼합물의 임의의 양을 제거하는 저속 벤치 톱 원심 분리기에서 미니 순간 펄스에 따라. </li><li> PCR 튜브에 동의 장착 된 탁상용 열 흔드는 개별적으로 표지 된 PCR 튜브에로드 샘플. 24 시간 동안 37 ° C에서 1,000 rpm으로 샘플을 흔들어. </li><li> 이하의 (w / v)이고, PK 100 ㎍ / ml의 최종 농도 2 %의 최종 농도 sarkosyl 추가 흔들. 다이제스트 샘플 열 셰이커 1 시간 동안 37 ° C에서 1,000 rpm으로. NOTE : PRP C의 PK 소화의 샘플 변환 후 보조기구 sarkosyl의 추가. 시드 화 샘플 (4.5 단계)에서 거짓 긍정적 인 신호는 거의 sarkosyl의 첨가에 의해 제거된다. </li><li> 17 immunoblotting에 다음 SDS-PAGE (16)에 의해 각 샘플에 남아있는 PRP 입술을 5 분 동안 95 ° C로 SDS-PAGE 샘플 버퍼, 열 샘플을 추가하고 해결. 주 : 시료 완충액의 첨가 및 가열은 샘플 즉시 처리 될 수 있거나 샘플을 -20 ° C에서 저장 될 수있다 또는 -80 ° C에 선행 면역 후. 여기에 제시된 모든 데이터는 NOVEX 누 페이지 젤 및 전송 시스템을 사용하여 생성되었다. CER 분석에서 개별 샘플은 샘플 버퍼 및 제조자의 지시에 따라 환원제로 처리 하였다. 이어서 샘플을 5 분 동안 95 ° C로 가열하고, 각 시료의 30 μl의 12 % 비스 - 트리스 프리 캐스트 겔에 로딩 하였다. </li><li> 척도로서 변환 효율 비 (CER)을 계산종 장벽. 농도계 (17)에 의해 PRP 해상도의 측정 수준과는 pH가 3.5 샘플의에 의해 pH가 7.4 시료의 전체 밀도를 나눕니다. 백분율을 생성하기 (100)에 의해 곱. </li><li> 주어진 TSE 에이전트 종의 평균 CER ± 표준 편차를 생성하기 위해 독립적 인 실험 반복 실험에서 데이터를 컴파일합니다. </li></ol>

<ol>
	<li>Colby, D. W., Prions Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harb Perspect Biol. 3 (1), a006833 (2011).</li><li>Hill, A. F., Collinge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+strains+and+species+barriers.">Prion strains and species barriers.</a> Contrib Microbiol. 11, 33-49 (2004).</li><li>Scott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+hamster+prion+protein+produce+species-specific+scrapie+infectivity+and+amyloid+plaques.">Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.</a> Cell. 59 (5), 847-857 (1989).</li><li>Prusiner, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenetic+Studies+Implicate+Interactions+between+Homologous+Prp+Isoforms+in+Scrapie+Prion+Replication.">Transgenetic Studies Implicate Interactions between Homologous Prp Isoforms in Scrapie Prion Replication.</a> Cell. 63, 673-686 (1990).</li><li>Telling, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+Propagation+in+Mice+Expressing+Human+and+Chimeric+Prp+Transgenes+Implicates+the+Interaction+of+Cellular+Prp+with+Another+Protein.">Prion Propagation in Mice Expressing Human and Chimeric Prp Transgenes Implicates the Interaction of Cellular Prp with Another Protein.</a> Cell. 83, 79-90 (1995).</li><li>Hill, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+prion+strain+causes+vCJD+and+BSE.">The same prion strain causes vCJD and BSE.</a> Nature. 389 (6650), 448-450 (1997).</li><li>Torres, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+modulating+the+prion+species+barrier+and+its+passage+consequences.">Elements modulating the prion species barrier and its passage consequences.</a> PLoS One. 9 (3), e89722 (2014).</li><li>Groschup, M. H., Buschmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+prion+diseases.">Rodent models for prion diseases.</a> Vet Res. 39 (4), 32 (2008).</li><li>Korth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abbreviated+incubation+times+for+human+prions+in+mice+expressing+a+chimeric+mouse-human+prion+protein+transgene.">Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.</a> Proc Natl Acad Sci U S A. 100 (8), 4784-4789 (2003).</li><li>Kocisko, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+specificity+in+the+cell-free+conversion+of+prion+protein+to+protease-resistant+forms:+a+model+for+the+scrapie+species+barrier.">Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.</a> Proc Natl Acad Sci U S A. 92 (9), 3923-3927 (1995).</li><li>Raymond, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+molecular+barrier+limiting+susceptibility+of+humans,+cattle+and+sheep+to+chronic+wasting+disease.">Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.</a> EMBO J. 19 (7), 4425-4430 (2000).</li><li>Fernandez-Borges, N., de Castro, J., Castilla, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+the+transmission+barrier.">In vitro studies of the transmission barrier.</a> Prion. 3 (4), 220-223 (2009).</li><li>Zou, W. Q., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidic+pH+and+detergents+enhance+in+vitro+conversion+of+human+brain+PrPC+to+a+PrPSc-like+form.">Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form.</a> J Biol Chem. 277 (46), 43942-43947 (2002).</li><li>Li, L., Coulthart, M. B., Balachandran, A., Chakrabartty, A., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+barriers+for+chronic+wasting+disease+by+in+vitro+conversion+of+prion+protein.">Species barriers for chronic wasting disease by in vitro conversion of prion protein.</a> Biochem Biophys Res Commun. 364 (4), 796-800 (2007).</li><li>Morawski, A. R., Carlson, C. M., Chang, H., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+prion+protein+conversion+suggests+risk+of+bighorn+sheep+(Ovis+canadensis)+to+transmissible+spongiform+encephalopathies.">In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies.</a> BMC Vet Res. 9, 157 (2013).</li><li>Chandler, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encephalopathy+in+mice+produced+by+inoculation+with+scrapie+brain+material.">Encephalopathy in mice produced by inoculation with scrapie brain material.</a> Lancet. 1 (7191), 1378-1379 (1961).</li><li>Browning, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prions+from+mule+deer+and+elk+with+chronic+wasting+disease+to+transgenic+mice+expressing+cervid+PrP.">Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.</a> J Virol. 78 (23), 13345-13350 (2004).</li><li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+sheep+and+goats+with+transmissible+mink+encephalopathy+virus.">Experimental infection of sheep and goats with transmissible mink encephalopathy virus.</a> Can J Vet Res. 51 (1), 135-144 (1987).</li><li>Rubenstein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+surveillance+and+antigen+conformation+determines+humoral+immune+response+to+the+prion+protein+immunogen.">Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.</a> J Neurovirol. 5 (4), 401-413 (1999).</li><li>Castilla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+species+barrier+by+PrP(Sc)+replication+in+vitro+generates+unique+infectious+prions.">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134 (5), 757-768 (2008).</li><li>Kubista, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27, 95-125 (2006).</li><li>Atarashi, R., Sano, K., Satoh, K., Nishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quaking-induced+conversion:+a+highly+sensitive+assay+for+prion+detection.">Real-time quaking-induced conversion: a highly sensitive assay for prion detection.</a> Prion. 5 (3), 150-153 (2011).</li><li>Ladner-Keay, C. L., Griffith, B. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaking+Alone+Induces+De+Novo+Conversion+of+Recombinant+Prion+Proteins+to+beta-Sheet+Rich+Oligomers+and+Fibrils.">Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to beta-Sheet Rich Oligomers and Fibrils.</a> PLoS One. 9 (6), e98753 (2014).</li><li>Mathiason, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+domestic+cats+to+chronic+wasting+disease.">Susceptibility of domestic cats to chronic wasting disease.</a> J Virol. 87 (4), 1947-1956 (2013).</li><li>Seelig, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

이 프로토콜의 성공적인 완료를 들어, 관심 기판 제조 (단계 2.1.2) 및 전환 반응 (단계 4.4.2)에 대한 비율을 기판에 시드에 사용되는 감염되지 않은 뇌 조직에서 PRP C 수준으로 지불해야합니다. 우리의 경험에 비추어 볼 때, 뇌는 한 PRP C는 면역 (그림 4)에 의해 존재로, 상당한 기간 부검 후 CER 기판으로 사용하기 위해 추출 할 수 있습니다. 실제로 일부는 자기 분해 CER 연?...

전염성 해면상 뇌증 (TSEs, 프리온 질환) 동물과 인간의 다양한 영향을 확장 배양 기간이 치명적인 신경 퇴행성 질환의 그룹입니다. TSEs의 추정 병인 제는 감염, 질병 - 관련에 PRP PRP (C)의 정상 세포 형태의 템플릿 기반의 변환에 의해 자기 전파 할 수있는 호스트 프리온 단백질 (PRP)의 미스 폴딩 이성체로 구성되는 감염된 호스트 1의 중추 신경계 조직에 축적 형태 PRP (TSE). 전염성 프리온은 일반적으로 포유 동물 종간 송신이 제한 이벤트 「TSE 종 장벽 &quot;이라는 개념이 탄생했다 종 특이 방식으로, 호스트 간에서 송신한다. 프리온 종 장벽의 생물학적 결정은 잘 이해되지 않습니다. 감염성 PRP TSE 호스트 PRP C C 사이의 아미노산 서열 유사성강력 변환 3-5 일어난다하지만, 생체 내 6,7에서 관찰 된 모든 프리온 전송 이벤트를 설명하기에 불충분 남아 있는지에 영향을 미친다. 따라서, TSE 종 장벽 특성은 주로 동물 도전 연구에 의존했다 : 서로 프리​​온에 주어진 종으로부터 나이브 동물을 노출시키고 질병 발병과 전송 효율의 지표로서 공격 속도로 얻어진 배양 시간을 측정. 이종 종에서 PRP C를 발현하는 마우스는 연구 8 이런 종류의에 사용됩니다. 트랜스 제닉 마우스의 제작 및 프리온 장기화 생물 검정뿐만 아니라 동물의 윤리적 사용과 관련된 비용은 TSE 종 장벽 실험 조사 장애물이다. TSEs에 대한 인간 종 장벽의 평가는 인간의 PRP C를 발현하도록 유전자 조작 된 생쥐에 의존한다. 이 마우스는 인간의 TSEs 또는이 t 수정에 굴복 긴 배양 기간을 필요로빠른 질병 발병 9 그가 인간의 PRP C 분자. 이 마우스에서 인간이 아닌 TSEs에 대한 배양 기간은 도전 부정적인 결과의 해석을 정상 마우스의 수명을 넘어 연장 할 수있다. 비 - 인간 영장류는 인간 종 장벽을 연구하기위한 프록시로서 사용되었지만, 그것은 인간 숙주에 진행됨에 따라 이러한 연구 정확하게 질병 요점을 되풀이되지 않을 동물 실험과 인간이 아닌 영장류의 다른 유형과 같은 문제점을 내포한다. 동물 생물 검정은 TSE에 종의 감수성을 측정하기위한 &quot;금 표준&quot;방법 남아 있지만, 장애물, 비용이 살아있는 동물 실험의 윤리 대안의 조사를 강요했다. PRP TSE에 의해 시드 단백질 분해 효소 K (PK)에 내성 상태 PRP (입술)에 호스트 PRP C의 변환을 평가에 따라 체외 분석,의 숫자는 개발 TSE 특검팀을 조사하기 위해 사용되어왔다ecies 장벽 10-12. 시험 관내 분석법의 예로는 무 세포 분석법 변환, 증폭 단백질 환상 (PMCA)를 미스 폴딩 및 변환 효율을 율 (CER) 10-14 분석을 포함한다. 이러한 분석 중 어느 것도 자연 감염 후 종 장벽에 관련된 계정 주변 요인을 고려하지 않지만, 모든 TSEs 잠재적으로 취약 호스트를 식별하는 데 유용 할 수 있습니다. 여기에서는 정상 뇌 균질 액으로부터 유도 두 변성 PRP C 기판이 벤치 탑 프리온 전환 반응에 사용되는 CER 분석 용 프로토콜, (도 1) (13)을 제시한다. pH를 7.4로 변성 기판 PRP C 만 종 장벽 (14)의 부재 하에서, 반응에 의해 시드 PRP TSE PRP 해상도로 전환시킬 수있다. 대조적으로, pH가 3.5에서 다른 기판에 PRP C 변성은 PRP 입술 다음 배양으로 변환 될 수 있도록어떤 종에서 PRP TSE로 변환을위한 제어 역할을합니다. pH가 3.5 기판의 pH가 7.4에 대해 기판의 PRP 입술에 PRP C의 변환의 비율은 종 장벽의 측정치를 제공한다. 우리는 CER 분석 다양한 TSEs에 실험실 쥐의 종 장벽 알려져을 예측 및 만성 소모성 질환 (CWD) 및 기타 TSEs 14 뿔양 등 다양한 포유 동물 종의 종 장벽을 예측하기위한 노력의 분석을 사용한 것을 발견했다 15. 유용이 방법을 찾을 수 TSE 종 장벽 또는 PRP C -to-PRP 해상도 변환의 평가를 신속하게 검사를 허용하는 도구에 관심 수사관.

<table><tbody><tr><td>12% Bis-Tris SDS-PAGE gels</td><td>Life Technologies</td><td>NP0342</td><td></td></tr><tr><td>0.5 mm Zirconium oxide beads</td><td>Next Advance</td><td>ZROB05</td><td>Other varieties of beads are also effective</td></tr><tr><td>Antibodies</td><td>various suppliers</td><td></td><td>Select appropriate primary and secondary antibodies for immunoblot detection of PrP&lt;sup&gt;res&lt;/sup&gt; from species of interest</td></tr><tr><td>Bead homogenizer</td><td>Next Advance</td><td>BBY24M</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>369434</td><td>High speed with temperature control</td></tr><tr><td>Conical tubes</td><td>any brand</td><td></td><td></td></tr><tr><td>Cotton-tipped applicator</td><td>Uline</td><td>S-18991</td><td></td></tr><tr><td>Cuphorn sonicator</td><td>Heat Systems-Ultrasonics</td><td>W-380</td><td>Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td></tr><tr><td>Densitometry software program</td><td>UVP</td><td>Vision Works LS Image Acquisition and Analysis software</td><td>Other programs, such as NIH ImageJ, will also work</td></tr><tr><td>Dounce homogenizer</td><td>Kimble Chase</td><td>885300</td><td></td></tr><tr><td>End-over-end mixer</td><td>Labnet International</td><td>H5600</td><td></td></tr><tr><td>Ethylenediaminetetra acetic acid</td><td>Boston Bioproducts</td><td>P-770</td><td>Hazardous chemical: eye irritation</td></tr><tr><td>Guanidine hydrochloride, 8 M</td><td>Thermo Fisher Scientific</td><td>24115</td><td>Hazardous chemical: acute toxicity, skin irritation, eye irritation</td></tr><tr><td>Heating block</td><td>Fisher Scientific</td><td>11-718</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>435570</td><td>Hazardous chemical: strong acid</td></tr><tr><td>Lithium dodecyl sulfate sample buffer, 4x</td><td>Life Technologies</td><td>NP0008</td><td>Hazardous chemical: skin &amp;amp; respiratory irritation, serious eye damage, flammable solid</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454-4</td><td>Hazardous chemical: acute toxicity, flammable liquid</td></tr><tr><td>Microcentrifuge tubes</td><td>any brand</td><td></td><td></td></tr><tr><td>Mini-centrifuge</td><td>Labnet International</td><td>C1301</td><td></td></tr><tr><td>N-lauroyl-sarcosine (sarkosyl)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L-5125</td><td>Hazardous chemical: acute toxicity, skin irritation, eye damage</td></tr><tr><td>Nonidet P-40</td><td>Amresco</td><td>M158</td><td>Hazardous chemical: skin irritation, eye damage</td></tr><tr><td>SDS-PAGE gel system</td><td>Life Technologies</td><td>NuPAGE electrophoresis system</td><td>Other SDS-PAGE systems will also work</td></tr><tr><td>PCR tubes (low-binding)</td><td>Axygen</td><td>PCR-02-L-C</td><td></td></tr><tr><td>Pestle homogenizer</td><td>Fisher Scientific</td><td>03-392-106</td><td></td></tr><tr><td>pH meter</td><td>Sentron</td><td>SI600</td><td></td></tr><tr><td>Polyvinyldifluoride membrane</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Proteinase K</td><td>Promega</td><td>V3021</td><td>Hazardous chemical: skin &amp;amp; eye irritation, respiratory sensitisation, organ toxicity</td></tr><tr><td>Reducing agent for SDS-PAGE samples, 10x</td><td>Life Technologies</td><td>NP0009</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D6750</td><td>Hazardous chemical: acute toxicity</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Thermo Fisher Scientific</td><td>28364</td><td>Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td>Hazardous chemical; strong base</td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>various</td><td>Use syringe size appropriate to volumes of substrate to be homogenized</td></tr><tr><td>Syringe needles</td><td>BD Biosciences</td><td>various</td><td></td></tr><tr><td>Thermoshaker (PCR tube shaker)</td><td>Hangzhou All Sheng Instruments</td><td>MS-100</td><td></td></tr><tr><td>Tris base</td><td>Bio Basic</td><td>77-86-1</td><td>Hazardous chemical: skin, eye, respiratory irritation</td></tr><tr><td>Triton X-100</td><td>Integra Chemical Company</td><td>T756.30.30</td><td>Hazardous chemical: acute toxicity, eye irritation</td></tr><tr><td>Vortexer</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr></tbody></table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

CER 분석의 성공적인 사용은 전환 반응에 사용 된 기판 쌍의 품질에 크게 의존한다. 이러한 이유로, CER 기판 쌍을 제조 절차에 따라, 품질 관리는 면역 블롯 (도 2)이 수행되어야한다. 양 기판의 경우, 분석 면역에 의해 10 ~ 25 μL. PRP C는 각각의 기판에 용이하게 검출 할 수 있어야하며 PRP C 수준은 둘 사이의 대략 같아야한다. 면역 반응성은 20 kDa의 위 주로 볼 수 있지만, ?...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

와 전염성 해면상 뇌증 종 장벽 평가<em&gt; 체외</em&gt; 프리온 단백질 변환 분석

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K 조회수.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

비디오: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Intraperitoneal 감염 후 면역 세포의 인신 매매 형광등 프리온 로즈 시간을 모니터링

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

연구

면역학 및 감염병학

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

보통 인간의 뇌에 용해와 불용성 PRP의 Oligomers의 절연

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

의학

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions의 단백질 Misfolding의 순환 증폭

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

조류 종의 소화 기관을 통과 한 후 감염성 프리온을 확인하기위한 절차

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

양에서 비정형 및 고전적인 스크레이키를 감지하는 임상 시험 프로토콜

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

실시간 떨게 유발 CWD Prions 지저분한 자료의 검색을 위한 변환 분석 결과

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Prion 안전 연구소 스와이프 테스트

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

면역조직화학에 의한 비정상적인 프리온 단백질 검출

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

신경과학

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

S. cerevisiae 에 단백질 기반 상속에 대 한 높은 처리량 검열

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

생물학

Detection of Misfolded Prion Protein Aggregates in Mouse Brain Tissue Using Western Blotting

Take tissue homogenate from a mammalian brain that contains both normal prion proteins and misfolded prion aggregates responsible for neurodegenerative diseases.
Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.
Treat the aggregates with a denaturing buffer at a high temperature to linearize the proteins and impart a uniform negative charge.
Load the sample onto an electrophoresis gel and separate the proteins into bands, which confirms the presence of aggregates.
Using an electric field, transfer the separated proteins onto a membrane.
Incubate the membrane in a blocking solution to mask non-specific binding sites.
Incubate with peroxidase enzyme-tagged antibodies, which bind specifically to the prion aggregates.
Wash the membrane to remove unbound antibodies, minimizing non-specific signals.
Add a chemiluminescent substrate and an oxidizing agent, enabling the peroxidase to oxidize the substrate and produce light.
Measure the light intensity to quantify misfolded prions in the homogenate.

와 전염성 해면상 뇌증 종 장벽 평가

요약

더 많은 비디오 탐색

이 비디오의 챕터