Bu çalışma, ABD Tarım Bakanlığı Hibe 1999-35212-8680 2.005-01.378 ve 2010-04288-01, USDA Formula Fonları ve Gürcistan&#39;ın Veterinerlik Tarımsal Araştırma Grant Devlet tarafından desteklenen olmuştur. Bu yayında açıklanan protokol yönettiği araştırma derslerin bir parçası olarak lisans öğrencileri tarafından kullanılmak için geliştirilmiştir: MIBO 4900L, GENE 4960H, BCMB 4960L, ve Georgia Üniversitesi BIOL 4960H ve Maurer birkaç moleküler epidemiyoloji araştırma projeleri öğrenciler tarafından kullanılan laboratuvar. Maurer ve Lee laboratuvarlar araştırmalar yapmış, pek çok lisans öğrencileri ve Bölüm Başkanı John Glisson araştırma ile lisans eğitimi bütünleştirmek için çabalarımızı desteklemek için teşekkür etmek istiyorum.

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. 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=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, 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=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

Çıkar çatışması ilan etti.

Salmonella cinsi (örn. invaziv) özgü hedefleri genlerin tespiti için piyasada bulunan PCR testleri. Ancak, tüm Salmonella insan ve hayvan popülasyonlarının prevalansı hastalığa neden yeteneklerini eşit. Salmonella LPS O-antijen ve flagellinH1 ve H2 antijenleri antijenik farklılıklar erken tanınması, bu antijenik farklılıklar üzerinde 2.500 serovars Salmonella farklılaşma yol açmıştır . Cinsi, Salmonella belirlenen 46 ayrı O antijenleri ve 86 ayrı flagellin (H) antijenleri vardır. Salmonella (H1) ya da iki farklı (H1, H2) flagellins sahip olabilir. O, H1, H2 antijenleri 2500 Salmonella serovars hesabı farklı bir birleşimi bugün kabul etti . Serovar belirlenmesi bireysel O ve H antijenleri elde edilen antijenik formül üzerinde olarak atanır. O antijenik formül olarak şöyle yazılabilir: H1: H2 ve burada &quot;-&quot; O-antijen için negatif Salmonella H1 veya H2 flagellin ve &quot;NT&quot; (tiplendirilemeyen) yokluğu anlamına gelir . Örneğin, bir S. serovar Typhimurium atanır 1,2 Enteritidis bir formülü D1 ile izole edilirken: i: enterica antijenik formül B ile izole g, m: -. Salmonella nüfusunun Bu nedenle PCR ile kolayca yararlanılabilir nükleotid düzeyde farklılıklar antijenik varyasyon dışa tezahürüdür. Biz belirlenmesi S. allelotyping PCR geliştirmek için belirli O ve H alleller ile ilişkili genetik farklılıkları tespit enterica serovars: Enteritidis, Hadar, Heidelberg ve Typhimurium (3). Hadar (C2: z10: e, n, x), Heidelberg (B-H1: Salmonella serovar Enteritidis H2 antijenik formüller (D1:: g, m) doğru atama ve raporlama O ile ilişkili tüm alleller için test : r: 1,2) ve Typhimurium (B: i: 1,2). O aleli veya antijen, H1 g bulgu bilgisi olmadan, tek başına m allel mutlaka Salmonella Salmonella serovars olarak Enteritidis olarak izole tanımlamaz: Agona, California ve Montevideo, aynı zamanda bu aynı allel sahip. Allelotyping PCR başlangıçta ön plana Salmonella serovars belirtilen algılamak için tasarlanmış iken, bu test bir ek 19 serovars (Tablo 3) tanımlayabilirsiniz. Bizim allelotyping PCR aslında O ve H allel tespit etmek için tasarlanmıştır. Pratikte ise, bize değil, O-spesifik antiserumlar kullanılarak izole edilen Salmonella kültürleri ile çalışırken O allelotyping PCR performans, O-antijen slayt aglütinasyon testi ile tespit etmek için daha pratik ve uygun maliyetli hale gelmiştir. Ancak, laboratuvar (2) bir ekran veya Salmonella FTA kartları (6) teslim izole yazmak için bu PCR kullanıyorsa O allelotyping PCR yöntemi tavsiye ve numunelerin ilk ekran ile Salmonella-spesifik PCR (4 include .) Allelotyping PCR PCR veya biyokimyasal / antijen testleri Salmonella olarak tanımlanır sadece bu örnekleri sınırlayın. Bu makalede açıklanan protokol Idaho Teknoloji Rapidcycler, bir çok ısıtma bloğu thermocyclers daha kısa sürede reaksiyon hacmi ve çalışan reaksiyon şartları küçültün sağlayan bir sıcak hava PCR ile kullanım için optimize edilmiştir. Ayarlayarak astar konsantrasyonları; veya MgCl 2 konsantrasyonları ayarlayarak bir ısıtma bloğu PCR ile kullanmak için bu protokol uyarlamak için tavlama sıcaklıkları düşürülmesi / yükseltilmesi deneysel reaksiyon koşulları optimize gerekebilir. Örneğin, MJ Research PTC-200 PCR aşağıdaki gibi protokol uyum sağlamak zorunda kalıyor. Tablo 1&#39;de açıklanan aynı reaksiyon hacmi ile çalışma, çalışma stok concentrationof i astar set 2.5 mcM seyreltilir, tavlama sıcaklığı 55 ° C - 45 ° C ve inkübasyon denatürasyon kez, tavlama ve uzatma adımları düşürülmüştür i / g, m allelotyping PCR için 20 saniye kadar uzatılmış oldu. Yayınlanan protokoller de dahil olmak üzere herhangi bir PCR için başlangıç ​​yukarı ve optimizasyonu, uygun pozitif ve negatif kontroller gereklidir. Hadar (C2: z10: e, n, x), Heidelberg (B: r - Enteritidis (D1:: g, m) Biz özellikle Anatum (E1:: e, h 1,6), bilinen Salmonella serovars elde önermektedir. : 1,2), Montevideo (C1: g, m, s: 1,2,7) ve Typhimurium (B: i: 1,2) ve bir laboratuvar Escherichia coli K12 suşu (DH5α LE393, JM109, vb . sırasıyla bu makalede anlatılan allelotyping PCR testleri pozitif ve negatif kontrolleri gibi). Bu Salmonella serovars birkaç test edildiği allel bağlı olarak, pozitif veya negatif kontrol olarak hizmet verecek. Bazı önlemler ve kurallar Bu ve diğer herhangi bir teşhis PCR yaparken takip edilmesi gerekir. İlk olarak, fiziksel ayırmaşablon hazırlama, PCR set-up ve jel elektroforezi mümkün PCR carry-over, kirlenme ve yanlış pozitif hatalı raporlama önlenmesinde kritik önem taşımaktadır. Ayrıca, bu kontroller olarak ortaya çıkan sorunları belirlemek ve sonuçları (5) yorumlanmasına yardımcı olarak gerekli kontrollerin dahil herhangi bir rutin PCR gereklidir. En önemlisi, tutarlılık Amplikon boyutu Amplikon (PCR ürünü) algılama andestimation elektroforetik koşulları açısından, özellikle önemlidir. Bu noktayı açıklamak için, biz bilerek Şekil 1A amaçlanan daha önceki elektroforez askıya aldı. Moleküler ağırlık standartları (şerit 1 ve 13) ve pozitif kontrol (2 şeritli), yeterince doğru boyutları tahmin ya da küçük boyutlarda farklılıklar vardır (~ 50 bp) DNA parçalarının ayrılması gözlemlemek için ayrılmış değildir. Yeterli DNA parçalarının ayrılması, özellikle moleküler ağırlık standartları, pozitif Amplikon boyutu ve bazen herhangi bir PCR (5 günlük çalışma gözlenen non-spesifik amplikonlarının ayırt &quot;gerçek pozitif&quot; doğru tahmin bağlıdır esastır .) Örneğin, Şekil 1B, şeritli 7 (boyut olarak ~ 700 bp) non-spesifik bir Amplikon vardır. Boya ön agaroz jel yarı yolda noktada sadece elektroforez, çok erken kesilmeli olsaydı, 500 bp ve 700 bp DNA parçalarının arasında çok az bir farklılaşma var olacaktır. Bu nedenle, örnek şeritli 7 yanlışlıkla i ve g, m allel hem de olumlu bildirilmiştir olurdu. Son olarak, herhangi bir test ile ilgili sınırlamaları tanımak gerekir. 1,5, 1,6, 1,7 antijen kompleksi; e, n, x / e H2 1,2 ait alleller ince genetik farklılıkları ayırt etmek H1/H2 allelotyping PCR çeşitli ayrımcı güç yok? n, g, m allel Z15 antijen içeren karmaşık ve H1 G antijen kompleksi,. Bir ya da iki farklı epitoplar bu flagellar antijenleri bulunan amino asit değişiklikleri oluşturmaktadır. Bu nedenle bize flagellin gen sekansı (3), bu bazen tek bir baz çifti farklılıklar yararlanabilir primerler dizayn için zordu. Örneğin, Salmonella serovars Dublin (D1: g: p) ve Berta (D1: f, g, t:) primerler mallelotyping, g da PCR pozitif. Bradford (1,5: r B), Heidelberg (1,2 B: r), Lagos (: i 1,5 B) H2 allelotyping primerler Typhimurium (1,2: i B) ayırt edemez Glostrup&#39;dan ikinci Winneba (B: r: 1,6), ya da Remo (C2: z10: e, n, Z15) (B: ​​r: 1,7), ya da Hadar (e, n, x C2: z10). Bu serovars karşılaşma ihtimalini de beraberinde getirir: Lagos, Bradford, Winneba, Remo veya Glustrup uzak bir olasılık ve ya testler sınırlama veya başka bir doğrulayıcı test feragatname sağlayan gerektirir. Sonuç olarak, bu PCR tabanlı serotipleme hızla S. tanımlar enterica serovars Enteritidis, Hadar, Heidelberg ve Typhimurium ve O, H1 ve H2 allel 19 ek serovars ile ilişkilidir. Bu testte serotipleme Salmonella geleneksel mikrobiyolojik yaklaşım hızlı, düşük maliyetli bir alternatif.

<table border="1"><tbody><tr><th> Malzeme Adı </th><th> Tip </th><th> Şirket </th><th> Katalog Numarası </th><th> Yorum </th></tr><tr><td> Luria Bertani </td><td></td><td> Balıkçı </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Mikrofuge&#39;de tüpler </td><td> 1.5 ml kapasiteli </td><td> Balıkçı </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Etanol </td><td> 200 kanıtı </td><td> Balıkçı </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> dH 2 O </td><td> Moleküler Biyoloji </td><td> Sigma </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak; PCR sadece) </td></tr><tr><td> 10 x PCR Buffer: </td><td> 20 mM MgCl 2 </td><td> Idaho Teknoloji </td><td></td><td> (Tampon BSA, Ficoll ve Tartrazin gelir) </td></tr><tr><td></td><td> 30 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td></td><td> 40 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td> PCR primerleri </td><td></td><td></td><td></td><td></td></tr><tr><td> DMSO </td><td></td><td></td><td></td><td></td></tr><tr><td> dNTP </td><td></td><td> Roche </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Taq DNA Polimeraz </td><td></td><td> Denville </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Kılcal pipetler </td><td> 10 ul hacmi </td><td> Idaho Teknoloji </td><td></td><td></td></tr><tr><td> Hızlı Cycler </td><td></td><td> Idaho Teknoloji </td><td></td><td></td></tr><tr><td> Agaroz </td><td> Düşük EEO, Moleküler Biyoloji </td><td> BioRad </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> 50 X TAE Tampon veya 5X TBE Tampon </td><td></td><td> Balıkçı </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Etidyum bromür </td><td></td><td> BioRad </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Yatay, dalgıç jel elektroforez jel kalıp odasına </td><td></td><td> BioRad </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Güç kaynağı </td><td></td><td> BioRad </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr><tr><td> Moleküler Imager Gel Doc Sistemi </td><td></td><td> BioRad </td><td></td><td> (Ya da herhangi bir karşılaştırılabilir tek bir kaynak) </td></tr></tbody></table> Tablo 4. Malzemeler

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

Bu cinse özgü Salmonella hedef genlerin belirlenmesi için mevcut ticari PCR testleri. Ancak, iki tür, altı alttür ve 2.500 &#39;in üzerinde farklı Salmonella serovars, ve tüm halk sağlığı için önemi eşit. Örneğin, S. bulma enterica alttür IIIa Arizona bir tablo yumurta katman çiftlikte S. izolasyonu ile karşılaştırıldığında önemsiz enterica alttür serovar Enteritidis, salmonelloz tablo yumurta tüketimi ile bağlantılı önde gelen nedenidir. Serovars antijenik lipopolisakkarid farklılıklar (LPS) (O antijen) ve flagellin (H1 ve H2 antijenleri) göre tespit edilir. Bu antijenik farklılıklar bu fenotip ile ilişkili genler ve gen allel çeşitliliğini dış görünüş. Multipleks PCR allelotyping geliştirilen dört ana S. arasında genetik farklılıkların tuşları enterica alttür I serovars kümes hayvanlarında bulunan ve ABD&#39;de önemli bir insan hastalığı ile ilişkili . PCR primer çiftleri, anahtar gen ya da belirli bir Salmonella serovar benzersiz ve bu gen ya da alleli için belirli bir büyüklüğe sahip bir Amplikon üretmek için tasarlanmış dizileri hedef alındı. Salmonella serovar özel LPS için PCR testi sonuçlarının kombinasyonu dayalı izole etmek için atanır ve flagellin allel gen. Bu makalede açıklanan multipleks PCR S. tespiti için özeldir enterica alttür I serovars Enteritidis, Hadar, Heidelberg, ve Typhimurium. Burada Salmonella izole serovar belirlemek için multipleks PCR nasıl kullanılacağını göstermektedir.

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, ve Typhimurium hızlı tespiti için bir multipleks PCR açıklar. Belirli Salmonella serovars, belirli bir serovar O-antijen biyosentezi küme ve flagellin benzersiz genler ve dizileri bir multipleks PCR hedef tespit edilebilir. Serovar hedef allel ilgili özel, boyut amplikonlarının (PCR ürünü) görünümünü dayalı izole Salmonella sonra atanır.

Allelotyping belirlenmesi için bir PCR Salmonella enterica Serovars Enteritidis, Hadar, Heidelberg ve Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

an-allelotyping-pcr-for-identifying-salmonella-enterica-serovars

Research

JoVE Journal

Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

21.6K Görüntüleme.  University of Georgia. Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, ve Typhimurium hızlı tespiti için bir multipleks PCR açıklar. Belirli Salmonella serovars, belirli bir serovar O-antijen biyosentezi küme ve flagellin benzersiz genler ve dizileri bir multipleks PCR hedef tespit edilebilir. Serovar hedef allel ilgili özel, boyut amplikonlarının (PCR ürünü) görünümünü dayalı izole Salmonella sonra atanır.

Video: An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Kaposi sarkomu ile ilişkili Herpes (KSHV) Yol Açtığı düzensiz Genler belirlenmesi

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

İmmünoloji ve Enfeksiyon

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Bir<em&gt; In vitro</emİnsan Makrofaj Farklılaşma ve Polarizasyon heterojenliği Eğitim için&gt; Model

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Tümör Hedefli ölçülmesi Büyüme ve Gen İfade Dynamics<em&gt; S. Typhimurium</em&gt; Bakteri

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

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

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

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

Bir Kuş Türlerinin Sindirim Sistemi geçtikten sonra Bulaşıcı prions belirlenmesi için prosedürler

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

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Bulaştırmak için bir protokol<em&gt; Caenorhabditis elegans</em&gt; Ile<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Fenotipinin yüksek verim Deneyi<em&gt; Salmonella enterica</em&gt; Typhimurium Derneği, Invasion, ve Makrofagların Çoğaltma

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Modüle c-di-GMP sinyal İşte Küçük Moleküller Tarama için Yüksek verim Kur kurulması<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

bir Genotiplendirme<em&gt; Staphylococcus aureus</em&gt; Tarafından Ribozomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...