Biz Dr. Philippe Erbs FCU1 gen dizisi ile sunduğunuz için teşekkür etmek istiyorum. Bu çalışma, Ulusal Sağlık Enstitüsü, hibe AI 26.649 tarafından desteklenen oldu.

1. Entamoeba histolytica FCU Negatif Seçim Sistemi <ol><li> Vektör kontrol hattı (HM1 / pKT3M) ve deneysel hattı (HM1/pKT3M-FCU1), daha önce açıklanan teknikler 1,2 üretildi . Tohum T-25 ölçülü balona stok tüpleri çalışma altında hatları ve TYI-S-33, 3 orta uygun antibiyotik (12 mg / ml G418) ekleyerek seçimi korumak . Matara 37 büyüme 16-18 saat sonra% 70-80 konfluent olmalıdır ° C </li><li> Sıcak TYI-S-33 orta, 5-florositozin (5-FC, Sigma) taze bir 50mm stok solüsyonu hazırlayın. Vortex titizlikle birkaç dakika için tamamen çözmek için 5-FC. Filtre sterilize filtresi 0.22 mikron geçirilerek stok solüsyonu. </li><li> 3-5 dakika buz üzerinde şişeler yerleştirerek ve oda sıcaklığında 5 dakika boyunca 200xg santrifüj hücreleri toplamak Hasat trofozoitleri. Taze TYI orta pelletini tekrar ve hücreleri saymak. </li><li> Tipik bir deney tasarımı, son hacim 1 ml 48 plaka başına 0.5 x 10 4 hücreleri kullanır; ve her bir test durumu için üç nüsha tekrarlar . Buna ek olarak, floresan ölçümleri kolaylaştırmak için, her zaman noktası başına suşu için ayrı bir plaka kullanılır. </li><li> Her test 5-FC, konsantrasyon, üç nüsha halinde de her Tohum 0.5 x 10 4 hücreler. TYI orta antibiyotik seçimi basınç koruyun. Şimdi 5-FC stok solüsyonu ve her kuyuya trofozoitleri gereken miktarda ekleyebilirsiniz. </li><li> 37, anaerobik bir torba içinde tabak inkübe ° C </li></ol> 2. Seçim Verimliliğinin Belirlenmesi <ol><li> 2 mg / ml DMSO Hücre tracker yeşil CMFDA (Invitrogen) stok solüsyonu hazırlayın. Çalışma çözeltisi, stok serum serbest TYI ortamda 1000 kat sulandırmak yapmak için. </li><li> Orta trophozoite kültür plakaları tamamen kaldırın ve 150 mcL serum serbest TYI içeren CMFDA ile değiştirin. Devam az 37 inkübasyon ° C 1 saat. </li><li> Kuyulardan boya çözüm çıkarın ve SPECTRAmax M2 mikroplaka spectrofluorometer plaka okuma. Dalgaboyu 492 nm dalga boyunda ve 517 nm okuma floresans emisyon olmalıdır. </li><li> Test transfektanları karşı kontrol hücreleri için floresan değerleri karşılaştırın. </li><li> Her bir zaman aralığı plakaları okumaya devam edin. Her plaka uygun pozitif (TYI sadece) ve negatif (25 mg / ml hygromycin) kontrol kuyuları kullanın. </li></ol> 3. Temsilcisi Sonuçlar E. histolytica transfektanları kodon optimize edilmiş rekombinant FCU1 (Şekil 1) sağlam bir ifade gösterdi. Bu protokol E. seçici eleme sonuçları doğru takip edildiğinde 0.5 mM 5-florositozin varlığı FCU1 ifade histolytica hücreleri. Kontrol hücreleri, aksine 5-FC, 5 mM konsantrasyonu normalde kadar büyüyebilir ve 72 ve 96 saatleri arasında izdiham ulaşmak devam (Şekil 2-a). Tedavi edilen kuyuların bir mikroskop altında incelendi 48 saat FCU1 taşıyan hücreleri tamamen parçalanmış. Bunlar da örnekleri çok sayıda (Şekil 2-b) içeren kantitatif çalışmalarda kullanılan hayati, boya CMFDA ile boyanarak floresan azalma gösterir. FCU1/5-FC sistemi E. için etkili ve güçlü bir negatif seçim aracı histolytica trofozoitleri. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="Şekil 1" /> Şekil 1. E. Üretimi negatif seçilebilir işaretleyici ifade histolytica HM1 transfektanları (A) FCU1 rekombinant protein, anti-c Myc antikor (üst panel) kullanarak bir western blot tespit edildi . Beklendiği gibi, bir 42kDa bant değil sadece vektör kontrolü transfektanları FCU transfektanlarından toplam lizat (bir ok ile gösterilen) tespit edilebilir. Alt panel aynı leke yükleme kontrolü gibi anti aktin antikor probed gösterir. (B) kantitatif gerçek zamanlı PCR Sonuçları lgl4 kontrol etmek için normalize FCU1 spesifik mRNA ifadesi gösteriyor. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="Şekil 2" /> E. Şekil 2 in vitro ilaç duyarlılığı negatif seçilebilir işaretleyici ifade histolytica transfektanları (A) kontrol transfektanları ve (b) FCU1 ifade transfektanları 48 plaka kültür sağkalım eğrileri. Hücreleri artan konsantrasyonlarda varlığı yetiştiği prodrogu-5-florositozin; X-ekseni üzerinde gösterilir hücre sağkalım CMFDA boyama ile her bir zaman aralığı belirlendi. Y ekseni floresan birimleri temsil eder ve hücrenin hayatta kalan nüfusun doğrudan bir yansımasıdır. FCU1 ifade transfektanları 0.5mm prodrogu konsantrasyon, kontrol ile karşılaştırıldığında önemli bir duyarlılık gösterdiğini dikkat ediniz. Hücreleri com bu kuyulardan 120h zaman noktasında gözlendiilgili kontrol kuyuları (veriler gösterilmemiştir) mikroskop altında görülen konfluent büyüme için hazırlandık. Higro negatif kontrol olarak kullanılan 25μg / ml hygromycin gösterir.

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. Microbiol. 45, 656-660 (1998).</li><li>Erbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cancer+gene+therapy+by+adenovirus-mediated+transfer+of+a+bifunctional+yeast+cytosine+deaminase/uracil+phosphoribosyltransferase+fusion+gene.">In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.</a> Cancer Res. 60, 3813-3822 (2000).</li><li>Braks, J. A. M., Franke-Fayard, B., Kroeze, H., Janse, C. J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+positive-negative+selectable+marker+system+for+use+in+reverse+genetics+in+Plasmodium.">Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.</a> Nucleic Acids Res. 34, e39-e39 (2006).</li><li>Maier, A. G., Braks, J. A. M., Waters, A. P., Cowman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+selection+using+yeast+cytosine+deaminase/uracil+phosphoribosyl+transferase+in+Plasmodium+falciparum+for+targeted+gene+deletion+by+double+crossover+recombination.">Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination.</a> Mol. Biochem. Parasitol. 150, 118-121 (2006).</li></ol>

Bu video üretimi Bu makalede kullanılan bazı reaktifler üreten Sigma Aldrich, Inc tarafından sponsor oldu.

Entamoeba moleküler biyoloji araçları önemli gelişmeler olmasına rağmen genler 4 silmek veya bozmak için hiçbir geçerli yöntemleri vardır. Gen belirli ifade demonte küçük saç tokası RNA&#39;lar RNA 6 müdahale 5, kullanımı sağlanır ve bakteriyel ifade dsRNA 7 olmuştur. Ilgili hedef genlerin etkili Ancak bu yöntemler, pahalı kimyasalların, antibiyotiklere karşı sürekli seçimi ve zaman içinde meydana gelen reversion yüksek insidansı (Alicia Linford, kişisel iletişim) 8 nedeniyle sürekli doğrulama için ihtiyaç kullanımı da dahil olmak üzere çeşitli sınırlamalar var . DNA replikasyonu 9 kökeni için sıraya gereksinimi görünüşte yok gibi herhangi bir plazmid Entamoeba çoğaltmak, böylece bir kez transfekte, genellikle plazmid tedavi etmek zordur. Bu rekombinasyon ve gen nakavt deneylerde sağlam plazmid olası kullanımını sınırlar. Negatif seçim işaretleri rekombinant alt popülasyonlar ortadan kaldırmak için kullanılabildiği gibi yöntemler hedef geninin anahtar bileşenleri. Biz başarılı bir kodon ifade Entamoeba histolytica FCU1 gen optimize edilmiş ve potansiyel olumsuz seçilebilir marker olarak test edilmiştir. Bu gen füzyon, insan tümör hücreleri intihar gen terapisi için kullanılan ve RNA ve DNA sentezi 10 inhibisyonu sonucu toksik aşağı ürünler haline prodrogu 5-FC metabolize olmuştur . FCU1 son zamanlarda, başka bir protozoon parazit Plasmodium olumsuz bir seçilebilir marker olarak kullanılır olmuştur. FCU1 ifade Plasmodium berghei hücreleri 5-FC ile in vitro ve in vivo tedavisinde prodrogu yaklaşık 1000 kat daha duyarlı olduğu tespit edildi,% 99.9 &#39;u öldürdü 11 sıtma eritrosit aşamalı bir fare modelinde rekombinant parazitlerin bulaşmasını E. FCU1 ifade histolytica hücreleri P. gözlenen hassasiyeti aksine prodrogu 5-FC, sadece 30 kat artmış duyarlılık gösterdi berghei. Bunun için olası nedenleri toksik metabolitleri ile rekabet büyüme orta nükleotidlerin büyük havuz olabilir. P. hem berghei ve P. falciparum FCU1 işaretleyici hedef gen bozulması kullanılmıştır. çalışmalar 11,12. Biz homolog rekombinasyon kullanarak Entamoeba genom işlemek için hedefliyoruz. FCU1/5-FC negatif seçim sistemi doğrulama, bu amaca yönelik önemli bir adım.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

Entamoeba histolytica amebiyazis etkeni ve dünya nüfusunun% 10 kadar bozar. Gen ekspresyonu yukarı ve aşağı düzenleme etkin moleküler teknikler stabil bir şekilde devam plazmid transfeksiyon güveniyor. Bu Entamoeba virülans faktörleri anlayışımız artış varken, kapasite eksojen DNA genomu içine ters genetik deneyler, bu parazitin çalışmada önemli bir avantaj olacaktır sağlayacak entegre etmek. Bu organizma tarafından sunulan zorluklar homolog rekombinasyon olayları ve transfekte trofozoitleri episomal plazmid DNA tedavi zorluk seçmek için yetersizlik içermektedir. Eksojen DNA, iyi niyetli bir genomik entegrasyon olay meydana geldi trofozoitleri belirlenmesi önemli bir sorun yüksek bir arka planda daha sonra sonuçlanır. Biz olumsuz bir seçim sistemi, bir maya sitozin deaminaz ve urasil prodrogu 5-florositozin (5-FC) fosforibozil transferaz Chimera (FCU1) ve seçim transgenik ifade dayalı gelişme raporu. FCU1 enzim toksik 5-fluorourasil ve 5-fluorouridin-5&#39;-monofosfat E. toksik olmayan, 5-FC dönüştürür FCU1 ifade histolytica hatları 30 kat prodrogu kontrol suşu göre daha duyarlı olduğu tespit edildi.

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

Biz olumsuz bir seçim sisteminin gelişme raporu<em&gt; E. histolytica</em&gt; Transgenik kimerik protein ekspresyonu (FCU1) ve prodrogu 5-florositozin seçimi dayalı. FCU1 protein, maya sitozin deaminaz ve phosphoribosyltransferase urasil bir füzyon. FCU1, İfade sonuçlandı<em&gt; E. histolytica</em&gt; 5-florositozin karşı duyarlılık.

Negatif Seçilebilir Marker Geliştirme Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

development-of-a-negative-selectable-marker-for-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.4K Görüntüleme.  University of Virginia Health System. Biz olumsuz bir seçim sisteminin gelişme raporu E. histolytica Transgenik kimerik protein ekspresyonu (FCU1) ve prodrogu 5-florositozin seçimi dayalı. FCU1 protein, maya sitozin deaminaz ve phosphoribosyltransferase urasil bir füzyon. FCU1, İfade sonuçlandı E. histolytica 5-florositozin karşı duyarlılık.

Video: Development of a Negative Selectable Marker for Entamoeba histolytica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Entamoeba histolytica DNA ve tRNA Metiltransferaz Dnmt2 (Ehmeth) Enzim ile in vitro tRNA Metilasyon Testi

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

İmmünoloji ve Enfeksiyon

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Akım Sitometri tarafından Timik Pozitif ve Negatif Seçim incelenmesi

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Ana-Hücre RNA sentezi üzerinde Viral Enfeksiyon Etkisi Tedbir tıklayın Kimya Kullanımı

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Yapımcı Epitel Hasar Analizi<em&gt; Entamoeba histolytica</em&gt; Enfeksiyon

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Etiket-ücretsiz Biyosensör Teknolojisi kullanılması Meme Kanserinde İnsan Lökosit Antijen BİYOMARKÖRLERİN Algılama

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Viral Örneklerin Negatif Boyanması İçin Kapsüllerin Biyokontainment İçerisinde Kullanımı

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific ve karışık DFS70 desen bir roman hepatit-2 ELITE/DFS70 nakavt substrat ile birlikte ANA tarama sırasında eşzamanlı ayrım

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...