Junko Wada Biz onu mükemmel teknik yardım için teşekkür ediyoruz. Prof Akio Nomoto PVR-IgG2a için nazik bir bakülovirüs ifade vektör sağlamak için minnettarız. Bu çalışma kısmen desteklenen Hibeler Yardım Polio Eradikasyon Tanıtım ve Araştırma, Sağlık, Çalışma ve Refah Bakanlığı Emerging Infectious Diseases ve yeniden ortaya çıkan.

1. Bakım ortamın hazırlanması (MM) ve büyüme ortamı (GM) MM ve GM hazırlanması için, 1 Çocuk Felci laboratuar kılavuzuna bakın lütfen. Dulbecco&#39;nun modifiye Eagle orta (DMEM),% 2 fetal buzağı serumu (FCS) ve% 10 FCS sırasıyla, MM ve GM yerine kullanılabilecek ile desteklenmiştir. 2. Anti-PV antikorların hazırlanması <ol><li> Anti-PV antikor (tip 1, 2 veya 3) Her flakon (vialde 0.5 ml) su ekleyerek sulandırın anti-PV antikor (RIVM PV yazarak antiserum). Bu sulandırılmış antikor çözümleri (her biri 0.5 mL), MM ayrı etiketli tüpler 4,5 ml ekleyin. Not: Bazı anti-PV antikor birçok sulandırılmış formu (vialde 0.5 mL) olarak verilmiştir. Anti-PV antikor böyle bir lot için, sulandırıldıktan için su eklenmesi gerekli değildir. </li><li> Sulandırılmış bir anti-PV antikor eşit miktarda karıştırın. Anti-PV1 +2, 2 +3 veya 3 +1: Mix her sulandırılmış antikor 1.8 mL (1.8 mL her bir karışım için + 1.8 ml = 3.6 ml). Anti-PV1 +2 +3: 1.2 mL (1.2 mL + 1.2 ml + 1.2 ml = 3.6 mL) her sulandırılmış antikor karıştırın. Not: PV örnekleri PV farklı serotipleri bir karışımını içerebilir. PV&#39;lerin karışımı ile örneklerinde PV tek serotip tanımlamak için gerekli olan, anti-PV antikorları (yani 1 +2, 2 +3, 3 +1, 1 +2 +3) havuzları. </li><li> FCS (0.72 ml) 1 / 5 birim, her anti-PV (1 +2, 2 +3, 3 +1, 1 +2 +3) antikoru (3.6 mL Her) (son 18.2% FCS) ekleyin. Not: FCS eklenmesi non-spesifik antikorların yol açtığı jelatin parçacığın aglütinasyon azaltılması için. </li><li> -20 ° C&#39;de PA tayininde kadar antikor havuzları tutun. </li></ol> 3. Sensitize jelatin parçacık, Sulandırma <ol><li> Sensitize jelatin parçacığın bir flakon (dondurularak kurutulmuş toz olarak verilmiştir) kiti dahil sulandırıldıktan tampon 1,5 ml ekleyin. </li><li> Nazik dokunarak iyice karıştırınız. </li><li> 4 sulandırılmış sensitize parçacık ° C tutun Sulandırılmış jelatin parçacık, 4 ° C&#39;de muhafaza en az 2 hafta aktif olabilir, ancak testin hemen önce hazırlanmalıdır. </li></ol> Önemli not: Sensitize jelatin parçacıklar olmayan aglütinasyon görüntü parçacıkların çok farklı olabilir, çünkü farklı sensitize jelatin parçacıklar çok karışmaz. Her deney için, tek bir çok hassaslaşmış jelatin parçacık kullanılmalıdır. 4. PV kimlik PA testi <ol><li> Bir mikro-titrasyon tabak hazırlayın. Bir virüs örneği tanımlanması için, 5 kuyu gerekir + en az bir negatif kontrol (aşağıda sensitize virüs ve antikor olmadan parçacık, iyi # 6 Örnek hiçbir virüs ve antikor kontrol bakınız). Toplam kuyu sayısı 5 x örnekleri tahlil + en az bir kontrol. Önemli not: Tayininde en az bir virüs ve antikor kontrol ayırın. Sensitize parçacık Bir flakon için yaklaşık 10 virüs örneklerinin belirlenmesi. </li><li> Mikro-titrasyon plaka 12 mcL / anti-PV antikor ya da GM ekleyin. (Aşağıdaki örneğe bakın) aşağıdaki gibi plaka düzenlemedir. Antikor (iyi # 1 ve 6) neden olmadan numunelerin, 12 GM mcL yerine anti-PV antikor ekleyin. Önemli not: Bu testte birçok PV örnekleri test edilmesi gerekir, çok kanallı pipet veya dağıtıcı Bu adım için faydalı olacaktır. <table border="1"><tbody><tr><td> Eh # 1 </td><td> Eh # 2 </td><td> Eh # 3 </td><td> Eh # 4 </td><td> Eh # 5 </td><td> Eh # 6 </td></tr><tr><td> GM </td><td> anti-P1 +2 +3 </td><td> anti-P1 2 </td><td> anti-P2 3 </td><td> ani-P1 3 </td><td> GM </td></tr></tbody></table></li><li> / Iyi virüs örneği 12 mcL ekleyin. Virüs olmayan bir örnek (iyi # 6), 12 GM mcL virüs çözümü yerine ekleyin. Not: Virüs örnek, enfekte olmuş hücreleri, genellikle L20B hücreleri veya RD hücre kültürü süpernatant. Önemli not: Birçok PV numuneleri bu testte test edilecek olursa, çok kanallı pipet, bu adım için faydalı olacaktır. <table border="1"><tbody><tr><td> Eh # 1 </td><td> Eh # 2 </td><td> Eh # 3 </td><td> Eh # 4 </td><td> Eh # 5 </td><td> Eh # 6 </td></tr><tr><td> virüs örneği </td><td> virüs örneği </td><td> virüs örneği </td><td> virüs örneği </td><td> virüs örneği </td><td> GM </td></tr></tbody></table></li><li> 4 ° C oda sıcaklığına kadar sulandırılmış sensitize parçacık çözümü alın ve hafifçe dokunun parçacıklar tamamen askıya almak. Önemli not: Nazik dokunarak parçacıkların tam süspansiyon sonra yağış asılı kalan parçacıkları önce plakaları, parçacıklar hemen eklendi olmalıdırte ve form homojen olmayan bir çözüm. </li><li> Tüm kuyuları sensitize parçacık / iyi 25 mcL ekleyin. Önemli not: Sensitize parçacık eklenmesinden sonra, plaka örnekleri tek tip tepki hemen başlatmak için karıştırma plaka tabi olmalıdır. Bu nedenle, hassaslaşmış parçacık ek plakasını örnekleri arasında bir zaman farkı en aza indirmek için dağıtıcı kullanarak yapılmalıdır. </li><li> Mikro-titrasyon plaka bir plaka mikser üzerine koyun ve 30 saniye için plaka karıştırın. </li><li> Oda sıcaklığında bir masanın üzerine beyaz kağıt üzerinde mikro-titrasyon plaka koyun (görsel gözlem kolaylığı için) (15 ~ 30 ° C) 2 saat Önemli not: Plaka sonuçları gözlem ve fotoğraf alma için tepkime sırasında ve sonrasında hareket etmeyin. Oluşan aglütinasyon stabil değildir ve kolayca hareketli plakalar rahatsız. </li><li> Plaka fotoğraflar çekin. Önemli not: Fotoğrafların Kısmi görünümü, sonuçların yorumlanması için sonraki muayene ve inceleme için yeterli veri veremiyordum. Bazı fotoğraflar, kuyulardan aglütinasyon görüntü odaklanarak plakaların tüm kuyuları kapsayacak şekilde alınmalıdır. </li></ol> 5. Sonuçların Yorumlanması <ol><li> Pozitif ve negatif kuyuların belirlenmesi. Hayır virüs ve antikor kontrolü (jelatin parçacıklar, non-aglütinasyon yağış) (Şekil 1 ve 2) bu örnekleri aglütinasyon karşılaştırın. Pozitif olarak değerlendirilecek hiçbir virüs ve hiçbir antikor kontrolü (non-aglütinasyon negatif kontrol) açıkça ayırt aglütinasyon Görüntüler ve aglütinasyon negatif kontrol için benzer görüntüler negatif olarak değerlendirilecektir. Açık, ancak sadece kısmi aglütinasyon gösteren Örnekleri &#39;Olumlu (kısmi aglütinasyon)&#39; gibi olumlu, ancak kayıt olarak değerlendirilecektir. </li><li> PV belirlenmesi. PV serotipleri belirlemek için anti-PV antikorları ile pozitif ve negatif kuyu dayalı sonuçları yorumlamak. Sonucun yorumlanması ilkesi hücre kültürü sistemi nötralizasyon testi (örneğin anti-P1 2 antikor için pozitif örnek tip 3 PV içerdiğini gösterir) &#39;e benzer. </li></ol> 6. Temsilcisi Sonuçlar PA testi PV kimlik Temsilcisi sonucu Şekil 1&#39;de gösterilmiştir. PV suşları sensitize jelatin partikülleri (No antikorlar örnekleri) aglütinasyon kurdu. Karşılık gelen anti-PV antikorları Hayır virüs benzer jelatin parçacıklar (non-aglütinasyon), yağış ve hiçbir antikorlar kontrolü (negatif kontrol) varlığında (örneğin, PV1 (Sabin) veya anti-P1 +2 varlığı gözlendi anti-P3 +1 antikorlar). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Şekil 1" /> Şekil 1. PV1 (Sabin) PA yöntemi ile PV belirlenmesi Virüs çözümleri, PV2 (Sabin) ve PV3 (Sabin) (1.2 x 10 8 CCID 50 / 12 mcL, 8.2 x 10 7 CCID 50/12 mcL virüs titreleri ve 3,5 x 10 7 CCID 50/12 sırasıyla mcL), PA yöntemi ile belirlenmesi için kullanılmıştır . Hayır virüs ve hiçbir antikorlar kontrolü jelatin partikülleri (non-aglütinasyon, Negatif kontrol) yağış gösterdi. <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Şekil 2" /> Şekil 2. Pozitif ve Negatif kuyu PV1 (Sabin) Virüs çözümleri, PV2 (Sabin) ve PV3 (Sabin) (2.4 x 10 8 CCID 50 / 25 mcL virüs titreleri, 1.7 x 10 8 CCID 50 / 25 mcL, yorumlanması. sırasıyla 7.3 x 10 7 CCID 50 / 25 mcL), 1 / 320 1 / 20 sulandırılmış, ve daha sonra seyreltilmiş örneklerinin 25 mcL anti-PV antikorları olmadan PA yöntemi ile incelendi.

<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>

Yeni Ürün Tasarımı Bölümü, Fujirebio Inc. Souji Masujima bir çalışanın

Bu PA testinin benzersiz bakış PV 2 tespiti için anti-PV antikor PVR molekülünün yerine kullanılmasıdır. Bu PV 3-6 üç serotipe düzgün bir yakınlığa sahip PV ile sensitize jelatin parçacıklar belirli bir etkileşim sağlar. PVR monomerik formları virion tek bağlayıcı site sadece orta yakınlık (~ -8 M 10 -7 asitlik sabiti) 7 çünkü, jelatin parçacıklar hassasiyete PVR immunoadhesin formu (PVR IgG2a) 8 ve başarıyla PV izolatların sensitiz...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Hassaslaşmış-jelatin parçacıklar </td><td> Fujirebio Inc. </td><td></td><td> Test kiti </td></tr><tr><td> Sulandırma tampon </td><td> Fujirebio Inc. </td><td></td><td> Test kiti </td></tr><tr><td> Microtitration plaka </td><td> Fujirebio Inc. </td><td></td><td> Test kiti </td></tr><tr><td> Anti-PV antikor </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

Küresel Polio Eradikasyon Girişimi, laboratuvar tanısı, akut flask paralizi (AFP) vakalarının dışkı örneklerinden izole edilip tanımlanması PV kritik bir rol oynamaktadır. Dünya Sağlık Örgütü (WHO) Küresel Polio Laboratuvarı Network, PV izolasyon ve tanımlama şu anda sırasıyla hücre kültürü sistemi ve gerçek zamanlı RT-PCR kullanılarak yapılmaktadır. PV-eradikasyonu sonrası çağda, basit ve hızlı bir şekilde tespit işlemleri ulusal laboratuvarlar çocuk felci vakalarının hızlı onay için yararlı olacaktır. Bu çalışmada, biz PV tanımlanması için geliştirilen roman PA testinin prosedürü gösterecektir. Bu PA testi PV reseptör (PVR) molekülü ve PV tüm serotipleri belirli ve düzgün bir benzeşme virion etkileşimi kullanmaktadır. İşlem son derece basittir (reaksiyon plakalar bir adım prosedürü) ve hızlı (sonuçlar reaksiyon 2 saat içinde elde edilebilir), ve sonuç görsel (jelatin parçacıkların aglütinasyon gözlem) görülmektedir.

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

Particle Agglutination Method for Poliovirus Identification

Poliovirus (PV) virüs alıcı molekül kullanan son zamanlarda geliştirilen bir roman partikül aglütinasyon (PA) tahlil hızlı ve kolay bir kimlik izin verdi. Bu makalede, biz PA PV kimlik tayini için prosedür gösterecektir.

Poliovirüs Kimlik Parçacık Aglütinasyon Yöntemi

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 Görüntüleme.  National Institute of Infectious Diseases. Poliovirus (PV) virüs alıcı molekül kullanan son zamanlarda geliştirilen bir roman partikül aglütinasyon (PA) tahlil hızlı ve kolay bir kimlik izin verdi. Bu makalede, biz PA PV kimlik tayini için prosedür gösterecektir.

Video: 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).

Belirlenmesi için 1.5 Saat Usulü Enterococcus</emDoğrudan Kan Kültürleri> Türler

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

İmmünoloji ve Enfeksiyon

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.

Fonksiyonel Motifler ve Bağlama Ortaklar peptid bazlı Kimlik

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 Virüs karşı Roman Antiviraller belirlenmesi için Tahliller

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.

Genler belirlenmesi için VIGS-aracılığı ile İleri Genetik Tarama konakçı olmayan Direnç ilgilendim


	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 kütle spektrometrisi kullanılarak kan kültür besi yerinden, Gram negatif bakterilerin hızlı belirlenmesi

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.

Jsubtomo kullanılarak Elektron Cryotomography Rekonstrüksiyonunda ikinci viral zarf glikoproteini Spikes ortalamasını

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.

Kapsüler Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Lateks Aglutinasyon tarafından

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.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

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.

Erken Viral Giriş Tespiti için Tahliller ve Antiviral Bileşiklerin Değerlendirilmesi

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.

PM10 parçacık bağlı Bet v 1 Allergen Akışı Sitometrik Analizi

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 ...