우리는 그녀의 뛰어난 기술 지원을 준코 와다에게 감사하고 있습니다. 우리는 친절 PVR - IgG2a위한 baculovirus 발현 벡터를 제공하는 교수 아키오 노모토에게 감사하고 있습니다. 이 연구에 의해 부분적으로 지원되었다 기금 - - 에이드의 소아 마비 박멸의 추진 및 후생 노동 장관의 전염병을 신흥 다시 새로운 연구하십시오.

1. 유지 보수 매체의 준비 (MM) 및 성장 매체 (GM) MM과 GM의 준비, 1 소아 마비 실험실 설명서를 참조하십시오. Dulbecco의 수정된 이글 배지 (DMEM)는 2퍼센트 소태아 혈청 (FCS)와 FCS가 각각 MM 및 GM의 대체로서 사용될 수 10 % 보충. 2. 안티 PV 항체의 준비 <ol><li> 안티 PV 항체 (타입 1, 2, 또는 3)의 각 유리병에 물 (유리병 당 0.5 ML)를 추가하여 Reconstitute 방지 PV 항체 (RIVM PV 입력 항혈청). 별도의 표시 튜브에 MM 4.5 ML이 재구성 항체 솔루션 (0.5 ML 각)를 추가합니다. 참고 : 안티 PV 항체 중 일부는 많은가 재구성 양식 (유리병 당 0.5 ML)로 제공됩니다. 안티 PV 항체의 같은 많은, reconstitution을위한 물 또한 필요하지 않습니다. </li><li> 재구성 방지 PV 항체의 동일한 음량을 섞는다. 에 대한 안티 PV1 2, 2 3, 또는 3 +1 : 혼합 각 재구성 항체의 1.8 ML (각 조합에 대해 1.8 ML + 1.8 ML = 3.6 ML). 에 대한 안티 PV1 2 세 : 혼합 각 재구성 항체 1.2 ML (1.2 ML + 1.2 ML + 1.2 ML은 = 3.6 ML). 참고 : PV 샘플은 PV의 다른 serotypes의 혼합물을 포함 수 있습니다. PVS의 혼합과 샘플의 PV 중 하나의 혈청형을 확인하려면 안티 PV 항체 (예 : 1 +2, 2 +3, 3 +1, 1 +2 +3)의 수영장이 필요합니다. </li><li> 각 백신 PV (1 +2, 2 +3, 3 +1, 또는 1 +2 +3) 항체 (3.6 ML 각) (최종 18.2 % FCS)에 FCS (0.72 ML)의 1 / 5 볼륨을 추가합니다. 참고 : FCS의 추가는 항체에 의한 젤라틴 입자가 아닌 구체적인 유착의 감소입니다. </li><li> -20 ° C에서 PA 분석에 사용할 때까지 항체 풀을 유지. </li></ol> 3. sensitized 젤라틴 입자의 Reconstitution <ol><li> sensitized 젤라틴 입자 중 하나 유리병 (동결 건조 분말로 제공)에 키트에 포함된 reconstitution 버퍼의 1.5 ML를 추가합니다. </li><li> 부드러운 감청에 의해 잘 믹스 </li><li> 4 재구성 sensitized 입자 ° C. 유지 재구성 젤라틴 입자는 4 ° C에 보관 적어도 이주 활성화 될 수 있지만, 단지 분석하기 전에 준비를해야합니다. </li></ol> 중요 사항 : sensitized 젤라틴 입자의 응집이 아닌 이미지는 입자의 많은에 의해 다를 수 수 있기 때문에, sensitized 젤라틴 입자의 다른 많은 혼합하지 마십시오. 각 실험, sensitized 젤라틴 입자의 단일 많이 사용해야합니다. 4. PV 식별 PA 분석 <ol><li> 마이크로 적정 플레이트를 준비합니다. 한 바이러스 샘플의 식별을 위해, 우리는 5 웰스 필요 + 적어도 하나의 대조군 (아래에 바이러스 및 항체없이 sensitized 입자 잘 # 6. 예에 대한 바이러스 및 항체 컨트롤을 참조하지 않음). 웰스의 총수는 분석에 + 하나 이상의 컨트롤 5 X 샘플입니다. 중요 사항 : 분석에 적어도 하나의 어떤 바이러스 및 항체 제어권을하시기 바랍니다. sensitized 입자 중 하나 유리병 10 바이러스 샘플을 식별 약입니다. </li><li> 마이크로 적정 플레이트 12 μL / 음 방지 PV 항체이나 GM의을 추가합니다. (아래 예제 참조) 다음과 같이 접시의 배열입니다. 항체 (물론 # 1 6)없이 샘플에서, 12 GM의 μL 대신 방지 PV 항체를 추가합니다. 중요 사항 : 많은 PV 샘플이 분석에서 테스트해야하는 경우, 멀티채널 피펫 또는 디스펜서이 단계에 대한 도움이 될 것입니다. <table border="1"><tbody><tr><td> 웰 # 1 </td><td> 웰 # 2 </td><td> 그럼 # 3 </td><td> 웰 # 4 </td><td> 웰 # 5 </td><td> 웰 # 6 </td></tr><tr><td> GM </td><td> 안티 P1 2 3 </td><td> 안티 - P1이 </td><td> 안티 P2 3 </td><td> 애니 - P1의 세 </td><td> GM </td></tr></tbody></table></li><li> 12 μL / 웰의 바이러스 샘플을 추가합니다. 바이러스없이 한 샘플 (물론 # 6)에서, 12 GM의 μL 대신 바이러스 솔루션을 추가합니다. 참고 : 바이러스 샘플 감염된 세포, 보통 L20B 셀 또는 RD 세포의 문화 뜨는입니다. 중요 사항 : 많은 PV 샘플이 분석에서 테스트해야하는 경우, 멀티채널 피펫이 단계에 대한 도움이 될 것입니다. <table border="1"><tbody><tr><td> 웰 # 1 </td><td> 웰 # 2 </td><td> 그럼 # 3 </td><td> 웰 # 4 </td><td> 웰 # 5 </td><td> 웰 # 6 </td></tr><tr><td> 바이러스 샘플 </td><td> 바이러스 샘플 </td><td> 바이러스 샘플 </td><td> 바이러스 샘플 </td><td> 바이러스 샘플 </td><td> GM </td></tr></tbody></table></li><li> 실내 온도 ° C 4에서 재구성 sensitized 입자 솔루션을 가지고, 천천히 완전히 입자를 중지 누릅니다. 중요 사항 : 부드러운 감청에 의한 입자의 완전한 정지 후, 입자가 정지 입자 precipita 전에 접시에 즉시 추가되어야테와 형식이 아닌 단일 솔루션입니다. </li><li> 25 μL / 음의 모든 우물에 sensitized 입자를 추가합니다. 중요 사항 : sensitized 입자를 추가 후, 접시는 즉시 샘플 균일한 반응을 시작하는 혼합 플레이트를 받게됩니다. 따라서, sensitized 입자의 추가는 접시에있는 샘플 간의 시간 지연을 최소화하기 위해 디스펜서를 사용하여 수행되어야합니다. </li><li> 접시 믹서에 마이크로 적정 판을 넣어 30 초에 대한 번호판을 섞는다. </li><li> 상온에서 테이블에 흰 종이에 마이크로 적정 플레이트를 (시각 관찰 편의를 위해) 올려 (15 ~ 30 ° C) 2 H. 중요 사항 : 결과 관찰하고 복용 사진에 대한 반응 중 후 접시를 이동하지 마십시오. 형성 응집이 안정되지 않으며 쉽게 접시를 이동하여 방해. </li><li> 접시의 사진을 찍어. 중요 사항 : 사진의 부분을 볼 해석에 대한 결과를 나중에 심사 및 검토를위한 충분한 데이터를 얻을 수 없습니다. 여러 사진이 우물의 응집 이미지에 초점을 접시의 모든 우물을 커버로 이동해야합니다. </li></ol> 5. 결과의 해석 <ol><li> 긍정적이고 부정적인 우물의 식별. 아니오 바이러스 및 항체 제어 (젤라틴 입자가 아닌 응집의 강수량) (그림 1 및 2)의로 샘플의 응집을 비교합니다. 분명하게 노 바이러스없이 항체 제어 (비 응집, 대조군)에서 구별 아르 응집의 이미지는 긍정적인 판단되어야하고, 대조군과 유사한 유착의 이미지는 부정적으로 판단해야합니다. 분명하지만 일부 분만 응집을 보여 샘플은 &#39;긍정적인 (일부 응집)&#39;로 긍정하지만, 기록으로 판단 될 수 있습니다. </li><li> PV의 식별. PV의 serotypes를 식별하는 안티 - PV 항체와 긍정적이고 부정적인 우물에 따라 결과를 해석한다. 결과의 해석 원리는 세포 배양 시스템에서 중화 시험 (예 : 긍정적인 안티 - P1이 항체에 대한 샘플 타입 3 PV가 포함되어 있음을 나타냅니다)의 유사합니다. </li></ol> 6. 대표 결과 PA 분석 PV 식별 대표 결과는 그림 1에 표시됩니다. 모든 PV의 변종이 sensitized 젤라틴 입자 (노 항체 샘플)의 응집을 형성. 해당 안티 PV 항체, 아니 바이러스와 유사한 젤라틴 입자 (비 응집)의 강수량없이 항체 제어 (대조군)의 면전에서 (예를 들어, PV1 (사빈) 안티 - P1이의 존재 또는 관찰되었다 안티 P3 1 항체). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="그림 1" /> 그림 1. PV1 (사빈)의 PA 분석하여 PV의 식별. 바이러스 솔루션, PV2 (사빈) 및 PV3 (사빈) (1.2 X 10 8 CCID 12분의 50 μL, 8.2 X 10 7 CCID 12분의 50 μL의 바이러스 titers, 그리고 3.5 X 10 7 CCID 12분의 50 각각 μL가) PA 분석하여 식별을 위해 사용되었습니다. 대한 바이러스없이 항체 제어 젤라틴 입자 (비 응집, 대조군)의 강수량을 보여 않습니다. <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="그림 2" /> 그림 2. 긍정적이고 부정적인 우물의 해석. PV1 (사빈)의 바이러스 솔루션, PV2 (사빈) 및 PV3 (사빈) (2.4 X 10 8 CCID 25분의 50 μL의 바이러스 titers, 1.7 X 10 8 CCID 25분의 50 μL와 각각 7.3 X 10 7 CCID 25분의 50 μL가) 320분의 1 to 20분의 1 희석되었고, 다음 희석 시료 25 μL는 안티 - PV 항체없이 PA 분석을 면밀히 검토했다.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

Souji Masujima 새​​로운 제품 디자인학과, Fujirebio 주식 회사의 직원입니다

이 PA의 분석의 독특한 점은 PV 2의 검출을위한 안티 - PV 항체 대신 PVR 분자의 활용이다. 이것은 PV 3-6의 세 serotypes에 균일 친화력과 PV와 sensitized 젤라틴 입자의 구체적인 상호 작용을 허용합니다. PVR의 monomeric 형태 virion에서 단일 바인딩 사이트에만 중간 동질성 (~ -8 M 10 -7의 해리 상수) 7을 가지고 있기 때문에 우리는, 젤라틴 입자의 sensitization에 대한 PVR의 i...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> Sensitized - 젤라틴 입자 </td><td> Fujirebio 주식 회사 </td><td></td><td> 테스트 키트 </td></tr><tr><td> Reconstitution 버퍼 </td><td> Fujirebio 주식 회사 </td><td></td><td> 테스트 키트 </td></tr><tr><td> Microtitration 플레이트 </td><td> Fujirebio 주식 회사 </td><td></td><td> 테스트 키트 </td></tr><tr><td> 안티 PV 항체 </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

세계 소아 마비 박멸 이니셔티브에서 실험실 진단 급성 이완된 마비 (AFP) 가지 경우 대변 샘플에서 PV를 분리하고 식별하여 중요한 역할을한다. 세계 보건기구 (WHO)의 글로벌 소아 마비 실험실 네트워크, PV 고립과 신원은 현재 각각 세포 배양 시스템 및 실시간 RT - PCR을 사용하여 수행되고있다. PV의 포스트 박멸의 시대에, 간단하고 신속한 식별 절차는 국가 실험실에서 소아 마비 가지 경우 신속한 확인 도움이 될 것입니다. 현재 연구에서 우리는 PV 식별용으로 개발된 새로운 PA 분석의 절차를 표시합니다. 이 PA의 분석은 PV 수용체 (PVR) 분자와 PV의 모든 serotypes에 구체적이고 균일 친화력이다 virion의 상호 작용을 활용합니다. 절차는 간단합니다 (반응 판에 한 단계 절차)와 빠른 (결과는 반응 2 H 내에서 얻을 수있다), 그리고 그 결과는 시각 (젤라틴 입자 응집의 관찰) 관찰합니다.

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

바이러스 수용체 분자를 이용한 최근에 개발된 새로운 입자 응집 (PA) 분석은 poliovirus (PV)의 신속하고 쉬운 식별을 허용했다. 이 문서에서는, 우리는 PV 식별을 위해 PA 분석을위한 절차를 표시합니다.

Poliovirus 식별을위한 입자 응집 방법

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Research

JoVE Journal

Immunology and Infection

15.2K 조회수.  National Institute of Infectious Diseases. 바이러스 수용체 분자를 이용한 최근에 개발된 새로운 입자 응집 (PA) 분석은 poliovirus (PV)의 신속하고 쉬운 식별을 허용했다. 이 문서에서는, 우리는 PV 식별을 위해 PA 분석을위한 절차를 표시합니다.

비디오: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

의 식별을위한 1.5 시간 절차 Enterococcus</em직접 혈액 배양에서> 종

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

연구

면역학 및 감염병학

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

기능 모티브과 바인딩 파트너 펩타이드 기반의 식별

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Bluetongue 바이러스에 대한 항 바이러스제 소설의 식별에 대한 분석

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

유전자의 식별을위한 VIGS - 중재 앞으로 유전학 검사는 Nonhost 저항에 참여


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo을 사용하여 전자 Cryotomography 재건에서 바이러스 성 봉투 ​​당 단백질 스파이크의 평균화

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; 라텍스 응집에 의해

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

초기 바이러스 성 항목 식별을위한 분석 실험 및 항 바이러스 화합물의 평가

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...