R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, ve P51OD011104: Yazarlar bu araştırmanın destek için aşağıdaki NIH ödül / subawards kabul etmek istiyorum.

2iD Görüntüleme Yazılımı ile 1. Kalibre Mesodissection Enstrüman 2iD görüntüleme yazılımı bu maddenin geri kalanı için yazılım veya program ya olarak adlandırılacaktır. Bu adımı doğru görüntüyü izlemek yanı sıra birden fazla slayt kareleri uyum için gereklidir. <ol><li> Enstrüman ve bilgisayarı açın. </li><li> Açık yazılım ve &quot;kalibrasyon aracı&quot; seçeneğini seçin. </li><li> Joystick&#39;i kullanarak sahne seyahat kalibre. Sol üst konuma aşamasına gider. Programda adım 1 altında basın &quot;set&quot;. </li><li> Doğru konumunu indirmek için sahneye taşımak. Programda adım 2 altında basın &quot;set&quot;. </li><li> İlk kafası düzeneğini yükseltmek için &quot;Z-tuşuna&quot; basarak aspirasyon kalibre. Joystick basın &quot;aspire darbe&quot; butonuna ve &quot;diseksiyon&quot; butonuna dalgıç kontrol çubuğu kadar üst pozisyona ulaşır. Programın 3. adımda altında basın &quot;set&quot;. </li><li> Basın &quot;aspirat sıfırlama &amp;# 8221; düğmesine basın. Piston 4. adımda alt pozisyon basın &quot;set&quot; durur kez. </li><li> &quot;Ölçek sekmesine&quot; üzerine tıklayın. </li><li> Boş tarafına kalibrasyon cetvel yerleştirin. </li><li> Gerekirse mikroskop odaklanın. </li><li> Fareyi kullanarak iki ölçek bar arasında bir çizgi çizin. </li><li> 2. adım. Basın &quot;hesaplama&quot; butonuna etiketli kutuda mesafeyi girin. </li><li> &quot;Crosshair&quot; sekmesine tıklayın. 1 motor hızını çevirin. </li><li> Kafa montaj içine xScisor yerleştirin. &quot;Z-ekseni&quot; düğmesine basarak z-ekseni hazır pozisyona alt kafa montaj. NOT: kafası düzeneği üst diseksiyon enstrüman üzerinde büyük bir yapıdır. XScisor bu maddenin geri kalanı için sarf değirmen biti olarak adlandırılacaktır. </li><li> Joystick düğmesine &quot;diseksiyon meşgul&quot; tıklayın. NOT: Bu düğme joystick üstünde yer almaktadır. </li><li> Görsel dönme merkezini tanımlayın. Bunu yapmak için, define ile dönme merkezi üzerinde crosshair merkezini taşımakProgramın adım 1&#39;de d parametresi. Crosshair hizalamak için piksel ayarlamak için &quot;x&quot; ve &quot;y ekseni&quot; barlarda yukarı ve aşağı okları kullanın. &quot;Done&quot; sekmesini tıklayın. </li><li> &quot;Ev&quot; simgesine tıklayın. NOT: Bu sağ üst köşesinde bulunan bir ev görüntüsü ile temsil edilir. </li></ol> 2.. Referans resim oluşturun <ol><li> Sahnede H &amp; E slayt yerleştirin. Slayt üstüne opak kapak yerleştirin. H &amp; E slayt istenen alana joystick&#39;i hareket ettirerek araç sürücü. Oluşturulan görüntü kaydetme. NOT: &quot;yakalama referans&quot; sekmesi veya &quot;teşrih&quot; sekmesi Ya bu adımı tamamlamak için kullanılabilir. </li></ol> 3. Diseksiyon için Doku hizalama <ol><li> Ana ekranda &quot;dokusu disseke&quot; seçeneğini tıklayın. </li><li> &quot;Operatörü&quot;, &quot;diseksiyon katılım numarası&quot;, &quot;referans erişim numarası,&quot; &quot;xScisor barkod numarası&quot; &amp; # doldurun8220; diseksiyon sıvı lot numarası, &quot;&quot; xScisor boyutu &quot;ve&quot; setup &quot;sekmesinde&quot; açıklama &quot;. </li><li> &quot;Doku bulmak&quot; sekmesine tıklayın. </li><li> İthalat önceden referans görüntü kaydedilir. </li><li> Sahnede 5 mikron kalınlığında bir boyanmadı FFPE slayt yerleştirin. Referans resimde gösterilen ile aynı alana sahne hareket ettirin. &quot;Insert image&quot; sekmesine tıklayın. </li><li> &quot;Hizalama&quot; sekmesine tıklayın. Boyanmamış görüntü referans görüntüyü hizalamak için fare ve ilişkili okları kullanın. </li></ol> 4. Slayt Annotate <ol><li> &quot;Annotatesekmesindeki&quot; sekmesine tıklayın. </li><li> Microdissected olacak slayt istenilen alanın etrafında bir daire çizmek için fareyi kullanın. </li></ol> 5.. Sarf Mill Bit yükleyin <ol><li> Bir sıvı hareket yukarı ve aşağı birkaç kez pistonu çekerek, burada istenen tampon, PKD tampon ile sarf değirmen biraz doldurun. Emin hava dışlamak olunkabarcıklar. </li><li> &quot;Z-ekseni butonuna&quot; basarak Z-ekseni kaldırın. </li><li> Makinenin içine sarf değirmen biraz yükleyin. Makinenin üst kısmına sarf değirmen biraz kaydırarak bunu böylece sarf değirmen bit siyah çizgi ile alet hatları üzerinde beyaz çizgi. Dalgıç doğru pozisyona indirdi izin vermek için basın &quot;aspirasyon reset&quot; düğmesine basın. NOT: Bu sarf değirmen biraz slayt kolay olmalıdır. Değilse, çentikler doğru hizada olduğundan emin olun. </li><li> Tekrar &quot;Z-ekseni&quot; düğmesine basarak alt Z-ekseni. </li></ol> 6.. Doku teşrih (Şekil 4) <ol><li> &quot;Teşrih&quot; sekmesini açın. </li><li> 1 motor ve aspirasyon hızları çevirin. </li><li> &quot;Show izleme&quot; kutusunu işaretleyin. Not: Bu, kullanıcının diseksiyon işlemi sırasında kesilmiş alan görselleştirmek için izin verir. </li><li> Teşrih etmeye hazır olduktan sonra, butonuna &quot;meşgul&quot; yanı sıra &quot;aspire &amp; # tutmaya devam8221; düğmesine doku incelemek için bir saat yönünün tersine joystick&#39;i hareket ederken. </li><li> Aspirat &quot;tam&quot; sekmesi kırmızıya döner kadar bir saatin tersi yönde incelemek için devam ediyor. </li></ol> 7. Sarf Değirmen biraz boş ve kaldır <ol><li> Sarf değirmen bitinin ucu altında bir 0.5 ul mikrofuge&#39;de tüp yerleştirin. </li><li> Basın &quot;aspirasyon&quot; kontrol çubuğu hızla mikrofuj tüpüne örnek çıkartmak için. </li><li> Kullanılmış sarf değirmen biraz çıkarmak için artan bir güçle basın. </li><li> Pistonu geri çekerek doku fragmanı durulayın. Sarf değirmen biraz içine kalan numuneyi ifade ileri pistonu itin. Doku parçası artık mikrofüj tüpü içinde ihtiva edilecektir. </li></ol> Isıl İşlem Ardından proteinaz K Sindirim 8. Lizleyin Örnek <ol><li> Doku örneği geri için proteinaz K içinde 5 ug ihtiva eden 70 ul TE pH 8.5 ekleyin. </li><li> Programlanabilir ısıtıcı-karıştırıcısı içine örneği yerleştir. </li><li> Aşağıdaki ayara göre programlanabilir ısıtıcı-çalkalayıcı kullanılarak tam sindirim: 1500 rpm&#39;de 30 dakika boyunca 60 ˚ ° C; 1500 rpm&#39;de, 15 dakika boyunca 82 ˚ ° C; ve 450 rpm&#39;de 1 dakika için 25 C ˚. Numune artık alt uygulamaları için hazır. </li></ol>

<ol>
	<li>Fend, F., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+pathology.">Laser capture microdissection in pathology.</a> J Clin Pathol. 53 (9), 666-672 (2000).</li><li>Esposito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+techniques:+laser+capture+microdissection--increasing+specificity+of+gene+expression+profiling+of+cancer+specimens.">Complementary techniques: laser capture microdissection--increasing specificity of gene expression profiling of cancer specimens.</a> Adv Exp Med Biol. 593, 54-65 (2007).</li><li>Adey, N., Bosh, D., Emery, D., Birch, L., Parry, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+Paraffin+Embedded+Slide+Mounted+Tissue+Sections.">Mesodissection of Paraffin Embedded Slide Mounted Tissue Sections.</a> Journal of Molecular Diagnostics. 14 (6), (2012).</li><li>Fleischmann, R. D., Alland, D., Eisen, J. A., Carpenter, L., White, O., Peterson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+comparison+of+Mycobacterium+tuberculosis+clinical+and+laboratory+strains.">Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.</a> J Bacteriol. 184 (19), 5479-5490 (2002).</li><li>Russell, D. G., Barry 3rd, C. E., Flynn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis:+what+we+don't+know+can,+and+does,+hurt+us.">Tuberculosis: what we don't know can, and does, hurt us.</a> Science. 328 (5980), 852-856 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+fibrous+tissue.">Mesodissection of fibrous tissue.</a> AvanSciBio. , (2013).</li><li>Paige, C., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penitentiary+or+penthouse+condo:+the+tuberculous+granuloma+from+the+microbe's+point+of+view.">Penitentiary or penthouse condo: the tuberculous granuloma from the microbe's point of view.</a> Cell Microbiol. 12 (3), 301-309 (2010).</li><li>Mehra, S., Alvarez, X., Didier, P. J., Doyle, L. A., Blanchard, J. L., Lackner, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granuloma+correlates+of+protection+against+tuberculosis+and+mechanisms+of+immune+modulation+by+Mycobacterium+tuberculosis.">Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.</a> J Infect Dis. 207 (7), 1115-1127 (2013).</li><li>Kaushal, D., Schroeder, B. G., Tyagi, S., Yoshimatsu, T., Scott, C., Ko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+immunopathology+and+mortality+despite+tissue+persistence+in+a+Mycobacterium+tuberculosis+mutant+lacking+alternative+sigma+factor,+SigH.">Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.</a> Proc Natl Acad Sci U S A. (12), 8330-8335 (2002).</li><li>Mehra, S., Kaushal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+reveals+extended+roles+of+the+Mycobacterium+tuberculosis+stress+response+factor+sigmaH.">Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor sigmaH.</a> J Bacteriol. 191 (12), 3965-3980 (2009).</li><li>Rohde, K. H., Veiga, D. F., Caldwell, S., Balazsi, G., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+the+transcriptional+profiles+and+the+physiological+states+of+Mycobacterium+tuberculosis+during+an+extended+intracellular+infection.">Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection.</a> PLoS Pathog. 8 (6), (2012).</li><li>Fontan, P. A., Voskuil, M. I., Gomez, M., Tan, D., Pardini, M., Manganelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mycobacterium+tuberculosis+sigma+factor+sigmaB+is+required+for+full+response+to+cell+envelope+stress+and+hypoxia+in+vitro,+but+it+is+dispensable+for+in+vivo+growth.">The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.</a> J Bacteriol. 191 (18), 5628-5633 (2009).</li><li>Rustad, T. R., Harrell, M. I., Liao, R., Sherman, 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=The+enduring+hypoxic+response+of+Mycobacterium+tuberculosis.">The enduring hypoxic response of Mycobacterium tuberculosis.</a> PLoS One. 3 (1), (2008).</li></ol>

Yazarlar herhangi bir mali çıkarları olduğunu ifşa.

Mesodissection patojenlerin geniş bir dizi neden olduğu patolojik lezyon slaytlardan RNA ekstre uygulanabilir bir tekniktir. Bu kullanıcı görüntü izler ve doğru olarak görüntü hizalayan bir zorunluluktur. Bunu gerçekleştirmek için, alet görüntüleme yazılımı yönleri aşağıdaki kalibre edilmelidir. Diseksiyon disseke ilgi slayt ve alan uyum yoksa, kullanıcı tekrar kalibrasyon gerekir. Diseksiyon sürecinde bir diğer kritik adım, diseksiyon önce (5.1 Adım) kabarcıklar yoksun böyle bir şekild...

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>. The corresponding author was changed from:
Teresa A. Hudock
to:
Deepak Kaushal

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>.

Erratum: A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Numuneler geleneksel elle bütün doku veya bir iğne ve neşter kullanılarak slaytlar birinden microdissected edilmiştir. Bu ilgi doku bölümünde ve çevre dokular bölümleri 2 arasında net bir ayrım gerektirir. Moleküler profil teknolojideki gelişmeler sayesinde, hücresel düzeyde doku değerlendirmek için artan bir ihtiyaç söz konusudur. Nedeniyle manuel mikrodisseksiyon sınırlamaları, LCM dahil teknikler daha fazla izolasyon hassasiyet sağlamak için kuruldu. Bu teknik, bir araştırmacı daha sonra, gen ekspresyonu profillemesi gibi alt-deneyleri için kullanılan çeşitli hücre tipleri ve slayt belirli hücre popülasyonlarının, izole sağlar. Alt deneyleri için çok etkili olsa da, LCM sınırlama olmadan değildir. Birincisi, LCM pahalı ve zaman alıcı bir süreçtir. Ayrıca nedeniyle RNA kararsız doğası, genellikle LCM örneklerinden 2 yüksek kalitede RNA elde etmek zordur. Nedeniyle diLCM sadvantages mikrodisseksiyon teknolojisindeki yeni gelişmeler hala bir ekonomik ve zaman hassas bir şekilde araştırmacıların daha fazla sayıda daha erişilebilir hale getirmek için gereklidir. Şu anda mevcut mikrodiseksiyon teknolojide Böyle bir gelişme mesodissection olarak bilinen bir tekniktir. Bu teknikte bir makine değirmen ilgi açıklamalı doku kesiti için kullanılan ve bir tüketim değirmen parça 3 içine emer. Daha sonra, bu numune bir toplama tüpüne aspire edilir ve alt uygulamaları için de kullanılabilir. Bu sistemin avantajı, belirgin olarak daha az pahalı ve zaman duyarlı olmasıdır. Bizim tecrübelerimize göre, sistem on dakika içinde bir akciğer granülom dokusu bölümünün diseksiyon sağlar. Öte yandan, geleneksel LCM olasılıkla işlemini tamamlamak için saat gerektirir. Sistem operatörü açıklama için bir karşılaştırma yanı sıra aracı olarak kullanmak için bir referans slayt yüklemenizi sağlar. Ayrıca bir rapor özetleyen inci oluşturulurdisseke slayt e alanı. Mesodissection iki ana dezavantajı vardır. Birden fazla hücre ayıklanması etkili olmasına rağmen, tek bir hücreyi izole etmek zordur. Buna ek olarak, oluşturulan görüntünün hassas diğer görüntüleme mikroskop kullanıyorsanız kadar net değildir. Tüberküloz (TB) önemli bir enfeksiyon hastalığı tüm dünyada insanlığın katil ve Mtb ile enfeksiyondan sonuçlanır. Mtb aerosollerle maruz kalan bireylerin çoğunda, latent enfeksiyon sınırlıdır. Yılda en az 10 milyon kişi, aktif verem hastalığı 4. sonuçlanır. Latent enfeksiyon esnasında, Mtb granülomlar olarak bilinen patolojik lezyonların içinde bulunur. Dolayısıyla Mtb enfeksiyon sonucu granülom 5 seviyesinde karar olduğu ileri sürülmüştür. Burada mesodissection Mtb neden granülomlarını microdissect için nasıl kullanılabileceğini göstermektedir. Kullanılan slaytlarEnfekte rhesus makakların FFPE akciğer dokusundan bulunmaktadır. Bu gösteri amaçla, kendi bütünlükleri içinde granülomlar teşrih edecek. Ayrıca RNA geri dokusundan elde edilebilir olduğunu göstermektedir. Bu teknik, çeşitli örneklerden doku örnekleri uygulanır ve daha sonra aşağı doğru bir çok analizde kullanılabilir.

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			<td height="20" style="height:20px;width:260px;">MeSectr</td>
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a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas

Diseksiyon bir on yıl için DNA, RNA ve protein düzeylerinde dokuların incelenmesi için kullanılmıştır. Lazer yakalama mikroskobu (LCM), bugün kullanılan en yaygın mikrodisseksiyon tekniktir. Bu teknikte, bir lazer fokal dehidre doku bölümü 1 üzerinde uzanan bir termoplastik zarı eritmek için kullanılır. Doku kesiti bileşik daha sonra kaldırılır ve membran ayrılır. Bu tekniği, doku inceleme için başarılı bir şekilde kullanılabilir olmasına rağmen, zaman alıcı ve pahalı. Ayrıca, bu teknik kullanılarak prosedürlerin başarıyla tamamlanması böylelikle, bunun kullanımını sınırlayan, bir lazer kullanılmasını gerektirir. Mesodissection adlandırılan yeni daha ekonomik ve pratik bir yaklaşım mikrodisseksiyonla LCM tuzaklar olası bir çözümdür. Bu teknik, değirmen, bir slayt monte edilmiş doku numunesinden arzu edilen doku MESO-1/MeSectr sistemini kullanmaktadır eşzamanlı olarak arzu edilen bir doku örneği kurtarmak için sıvı dağıtma ve aspirasyon sırasındaBir sarf değirmen biraz. Diseksiyon işlemi başlamadan önce, kullanıcı (FFPE) gömülü formalin sabit parafin (H &amp; E) referans slayt lekeli bir hematoksilen ve eozin ile slayt hizalar. Bundan sonra, operatör, arzu edilen diseksiyon alan annotates ve uygun bir bölümü incelemek için devam eder. Program diseksiyonu arşivlenmiş bir görüntü oluşturur. Mesodissection ana avantajı, bu deneyde nesil örnek kurmak on dakika ortalama alarak, bir slayt incelemek için gerekli kısa sürmesidir. Ayrıca, sistem önemli ölçüde daha düşük maliyetli ve kullanıcı dostudur. Hafif bir dezavantajı, bu lazer yakalama mikroskopisi gibi hassas olmasıdır. Bu yazıda mesodissection Mycobacterium tuberculosis (MTB) neden FFPE granülomlarında slaytları RNA ayıklamak için nasıl kullanılabileceğini göstermektedir.

Anılan protokol FFPE doku slaytlar RNA ayıklamak için yeni bir mesodissection tekniği nasıl kullanılacağını gösterir. Bu protokolün etkinliği çeşitli enfeksiyon aşamalarında Mtb ile enfekte NHP&#39;ın akciğer granülomlarının FFPE slaytlar aracılığıyla gösterilir. 1-3 alet. Şekil 4. görüntüleri Rakamlar diseksiyon sürecinin nasıl oluştuğunu gösteriyor ve yazılım. Tablo 1. tarafından oluşturulan çıkan gör...

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A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Diseksiyon yaygın doku içindeki DNA, RNA ve protein incelenmesi için kullanılmıştır. Lazer yakalama mikroskobu (LCM) en sık kullanılan yöntem, ancak, yeni bir freze tekniği, mesodissection, son zamanlarda kullanılabilir. Biz mesodissected formalin sabit parafin Mycobacterium tuberculosis granülomlarının gömülü doku slaytlar RNA çıkarma göstermektedir.

Kurtarma Bir Roman Mikrodiseksiyon Yaklaşımı<em&gt; Mycobacterium tuberculosis</emAkciğer granülomlara Gömülü Formalinle Sabit Parafin dan&gt; Özel Transkript

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

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Research

JoVE Journal

Immunology and Infection

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

7.9K Görüntüleme.  Tulane National Primate Research Center.  Diseksiyon yaygın doku içindeki DNA, RNA ve protein incelenmesi için kullanılmıştır. Lazer yakalama mikroskobu (LCM) en sık kullanılan yöntem, ancak, yeni bir freze tekniği, mesodissection, son zamanlarda kullanılabilir. Biz mesodissected formalin sabit parafin Mycobacterium tuberculosis granülomlarının gömülü doku slaytlar RNA çıkarma göstermektedir. 

Video: A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Numune Hazırlama<em&gt; Mycobacterium tuberculosis</emNükleer Manyetik Rezonans metabolomik Araştırmaları&gt; özler

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

İmmünoloji ve Enfeksiyon

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Büyüme Mycobacterium tuberculosis Biyofilmler

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNA Hedefleme Digoksin-etiketli DNA probu kullanılarak Parafine gömülmüş dokular içinde Bakterilerin in situ tespiti

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Fonksiyonel Araştırmaları 3D İnsan Akciğer Doku Modeli<em&gt; Mycobacterium tuberculosis</em&gt; Enfeksiyon

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Bir Yüksek verim Uyumlu Deneyi Makrofaj, pasajlamaya karşı ilaç etkinliğini değerlendirmektir<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...