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>

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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. 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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 border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

연구

의학

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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 ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

췌장 신경 가소성 시뮬레이션 :<em&gt; 체외</em&gt; 이중 신경 가소성 분석

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

도구로 아데노 관련 바이러스를 사용하여 질병 망막 장벽을 연구하는

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

신생아 저산소 - 허혈성 뇌증의 돼지 모델

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

미숙아의 모델링 뇌증은 흰쥐의 내부 - 양수 리포 폴리 사카 라이드와 태아 저산소증 - 허혈을 사용하여

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

항암 약의 특이성 평가<em&gt; 체외</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

신생아 쥐의 달팽이관 Explant 기법으로<em&gt; 체외에서</em&gt; 청력 연구의 스크리닝 도구

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...