Bu çalışmada, Halk Sağlığı Servisi hibe tarafından desteklenen AI-34431 Ulusal Alerji ve Bulaşıcı Hastalıklar Enstitüsü ve VA Tıp Araştırma Fonu (DAH) (DAH).

1. Cam slaytlar Leptospira fiksasyonu <ol><li> Leptospira interrogans BSL2 patojen bir sınıf. Canlı hücreler ile çalışma, eldiven, laboratuvar kat ve steril bir kaput pipetleme adımları uygun kullanım gerektirir. </li><li> 30 ° C&#39;ye ulaşana kadar orta-yaklaşık 6 gün geç-log fazı (5 x 10 8 hücre / ml 5 x 10 7 yoğunluğu)% 1 tavşan serumu ile desteklenmiş, EMJH medium11 Leptospira büyütün </li><li> Oda sıcaklığında 7 dakika için ~ 2000 xg&#39;de santrifüj kültür Hasat. </li><li> Aspirasyon ile süpernatantı alın ve yavaşça tekrar süspansiyon fosfat pelet nihai konsantrasyonu 5 x 10 8 hücre / ml salin (PBS) -5 mM MgCl 2, pH7.2 tamponlu. </li><li> Iki iyi oda cam slaytlar her bir kuyu için 1 ml hücre süspansiyonu ekleyin ve 30 ° C&#39;de 80 dk hücreleri uymak için izin verecek. </li><li> Aspirasyon ile bağlanmamış hücreleri içeren sıvı dikkatlice çıkarın. </li><li> 1 ml / PBS-5 mM MgCl 2% 2 paraformaldehid de ekleyerek, kalan sağlam bakteri cam slaytlar sabitleyin . 30 en az 40 dakika boyunca inkübe ° C. Bu slaytlar, yüzey maruz proteinleri değerlendirilmesi için. </li><li> Alt-yüzey proteinleri değerlendirmek kontrol slaytlar için, buz, soğuk metanol 1 ml / 100% ile tespit dış membran permeabilize. -20 ° C&#39;de 20 dakika inkübe edin. Metanol çeşitli şekillerde hareket eder, dış zar permeabilizes, proteinler denaturates ve cam slaytlar düzeltmeleri hücreleri. </li><li> Aspirasyon ile ajanları tespit çıkarın. </li></ol> 2. Spesifik antikorların ile Etiketleme <ol><li> 1 ml / engelleme tamponu (Difco Leptospira Zenginleştirme EMJH) ekleyerek non-spesifik bağlama engelleyin. 30 ° C&#39;de 90 dakika inkübe edin. </li><li> Spesifik antikor (bağışıklık tavşan sera ya da fare monoklonal antikorlar, OmpL54 10 için özel burada poliklonal tavşan serumu, FlaA1 12, ve OmpL1 13) ve herhangi bir antikor içeren ön-immün tavşan sera veya fare sofu sıvı (negatif kontrol olarak kullanılan) sulandırınız tampon engelleme. Her antikor için çözeltiler ampirik hücre antijen-antikor reaktivite ve bol miktarda protein, antikor titresi bağlı olarak belirlenmelidir. Her zamanki aralığı 1:600 ​​için 01:50. </li><li> Aspirasyon engelleme tamponu çıkarın ve 1 ml / seyreltilmiş primer antikor ekleyin. </li><li> 1s için 30 ° C&#39;de inkübe edin. </li><li> Aspirasyonu ile sıvı çıkarın ve kuyulardan PBS (1 ml / iyi) ile üç kez yıkayın. </li></ol> 3. Leptospires, görselleştirilmesi <ol><li> 1 ml / Alexa seyreltilmiş Fluor 488-etiketli sekonder antikor (keçi anti-tavşan IgG veya keçi anti-fare IgG ya) 1:2000 ve floresan nükleik asit leke, 4&#39;6-diamidino-2-fenil-indol dihidroklorür (Ekle DAPI) 0.25 mcg / ml tampon engelleme nihai konsantrasyonu seyreltilmiş. Bu adım, antikor bağlayıcı ve bağlayıcı sırasıyla antikor bağımsız tüm spiroketlerine varlığı algılama sağlar. </li><li> Slaytlar, 30 ° C&#39;de 45 dakika inkübe edin. </li><li> Aspirasyonu ile sıvı çıkarın ve distile su (1 ml / kuyu) kuyular PBS ile iki kez ve bir kez yıkayın. </li><li> Odaları ve cam slaytlar ve yaklaşık 10 dakika süreyle kurumaya yapışkan şeridi çıkarın. </li><li> Altın anti-solmaya montaj orta (slayt başına 2 x 20 ul) ve 24 x 50 mm kapak kayma uzatmak ekleyin. </li><li> Gece karanlıkta, oda sıcaklığında inkübe edin. Bu adım, montaj orta (sertleşmesine) tedavi etmek için izin için gerekli. </li><li> Oje ile kapak kayma Seal. </li><li> DAPI için mavi / mavi bir algılama filtresi ve Alexa Fluor 488 yeşil bir algılama filtresi kullanarak floresan mikroskobu ile boyama gözünüzde canlandırın. Numunelerin doğru bir temsili yapılmış olduğundan emin olmak için, her numune için tüm oda alanını değerlendirmek emin olun. </li><li> Görüntüleme, her örnek için bir temsilci alan verileri kaydedin. Görüntüleme Alexa Fluor 488 floresan, tüm örnekler için aynı pozlama süresi kullanın. Tutarlı bir pozlama süresi kullanma, test antijenlere karşı kontrolleri ile sonuçlarının daha doğru bir karşılaştırma sağlayacaktır. </li></ol> 4. Temsilcisi Sonuçlar: Leptospira interrogans hücreleri ile yüzey ve alt yüzey proteinlerine karşı antikorlar kullanılarak yüzey IFA sonuçlarının örnekleri Şekil 2&#39;de gösterilmiştir. Önemli floresan bir protein yüzey maruz kalmaktadır (Şekil 2A) faiz sağlam leptospires örneklerinde tespit edildiğinde testi pozitif olarak kabul edilir. Genellikle, bir pozitif test (burada OmpL54 yüzey maruz kalan), yaklaşık aynı floresan bir yeraltı karşı antikor kullanarak dış membran bütünlüğü için bir negatif kontrol dahil etmek esastır (Şekil 2A), hem de sağlam ve permeabilize hücreler görülmektedir tercihen periplazmik, protein (örneğin leptospiral FlaA1 Şekil 2B). Veri dış zarı bozulmadan duri kalmıştır sadeceng deneyi, yani bir alt yüzey proteinine karşı antikorların sağlam hücreleri (Şekil olarak permeabilize örnek kıyasla floresan veya çok zayıf floresan. 2B) tepkimeye girmeyen, ilgi protein (ler) olduğu söylenebilir. yüzey maruz. Net veriler için ek bir negatif kontrol olarak önceden bağışıklık serumları (poliklonal antikor durumda) ile deneyler dahil edilmesi esastır ve bozulmamış veya permeabilize hücreleri (Şekil 2A, 2C) önemli floresan vermeyecektir. Leke DAPI olumsuz sonuçlar gözlenmiştir (no Alexa Fluor 488 floresan) leptospires görselleştirmek için gerekli. Propidium iyodür ile Counterstaining DAPI için kabul edilebilir bir bir alternatif. Bu yöntem, eşit bolluk diğer antijenlere göre tek bir antijen ama daha az yüzey maruz epitoplar kabul edilmesi antijenik epitoplar maruz birden fazla yüzeye bağlı olabilir nitel yerine parlak floresan gibi nicel. Bu nedenle floresan yoğunlukları farklı proteinler arasında sağlam karşı permeabilize hücreleri ve floresan değil şiddetleri aynı antikorlar, reaktivite karşılaştırmak için kullanılabilir. Bu ayrım, önemli ölçüde daha az floresan permeabilize hücreleri (Şekil 2C) daha sağlam hücrelerinde gözlenen olduğu belirsiz bir sonuç elde edilir önemlidir. Böyle bir sonuç ya da protein (sağlam hücreleri bazı non-spesifik bir arka plan boyama) bir alt-yüzey yeri olduğunu gösterir, antikorlar birden fazla olabilir tercihen metanol permeabilization veya bir protein sonra maruz kalan yerli olmayan epitoplar bağlayıcıdır lokalizasyon siteleri gibi başka bir spirochete, Borrelia burgdorferi 14 Erp ve Elp proteinler tanımlanmıştır. Her iki durumda da, bu gibi belirsiz bir IFA sonucu faiz protein yüzey maruz kalan ya da 10 olup olmadığını belirlemek için, yüzey biyotinilasyon veya yüzey proteoliz gibi alternatif yaklaşımlar gerektirir. <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="Şekil 1"/> Şekil 1. Leptospira interrogans spiroketlerine immuno-floresans test yüzey akış şeması. Birincisi, her iki iyi oda slaytlar iyi 1 ml hücre süspansiyonu eklendi (her bir deney için en az iki kaydıraklı) ve uymaları leptospires inkübe. Sonra, leptospires sağlam dış membran (OM) ve permeabilize OM örnekleri için 1 ml / 100% soğuk metanol ile numuneleri için 1 ml /% 2 paraformaldehid (PFA) ekleyerek odasına slaytlara sabitlenir. Sonra, leptospires 30 yeraltı proteinleri (İlköğretim Ab) ° C de 90 dakika boyunca kontrol antikorlar ile birlikte yüzey maruz proteinleri tanıyan antikorlar ile inkübe edilir. Daha sonra, primer antikorlar 1 ml / Alexa Fluor 488 konjuge sekonder antikor ile inkübasyon tarafından tespit edilir. Son olarak, hücreler floresan mikroskopi tarafından görüntülenmiştir. <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="Şekil 2"/> Şekil 2 leptospiral proteinler Yüzey immünofloresan analiz. Leptospira interrogans spiroketlerine bağışıklık ve ön-immün sera probed ve olumsuz sonuçlar spiroketlerine varlığını göstermek için bir DAPI counterstain ile eşleştirilmiş. Yeşil floresan filtre Alexa Fluor 488 hücre DNA bağlayıcı DAPI tespit maruz kalan proteinler ve mavi / camgöbeği floresan filtre yüzey bağlayıcı etiketli ikincil antikor tespit etmek için kullanılmıştır. A) Leptospires bir yüzey maruz protein, OmpL54 10 tanıyarak antikorlar araştırdı. B) Hücreler periplazmik yeraltı protein, FlaA1 (negatif kontrol) karşı antikorlar ile araştırdı. C) Hücreler leptospiral porin, OmpL1 karşı antikorlar ile araştırdı. Leptospires tavşan sera Cilt Alexa Fluor 488 konjuge keçi anti-tavşan IgG parçaları tespit edildi. Tüm görüntüler 4 sn uzun maruz kaldıktan sonra alınır.

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Bizim yüzey IFA tekniği çeşitli borrelial 15, 16 ve leptospiral 12, 17 proteinler yüzeye maruz göstermek için kullanılan benzer. Burada anlatılan yüzey IFA yöntemi cam slaytlara uygun Leptospira hücrelerinin eğilim yararlanarak dış membran bütünlüğünü korumak için bir çaba hücrelerin manipülasyonu en aza indirmek için tasarlanmış. Bu yöntem, dış membran stabilize tamponu (PBS 5 mM MgCl 2) ve daha az yıkama adımları ile yıkanarak paraformaldehid pro...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> İki iyi oda cam slaytlar </td><td> Lab-Tek </td><td> 177380 </td><td></td></tr><tr><td> Tavşan serumu </td><td> Rockland Immunokimyasallar </td><td> D209-00-0100 </td><td></td></tr><tr><td> Leptospira Zenginleştirme EMJH </td><td> BD Difco </td><td> 279510 </td><td></td></tr><tr><td> Alexa Fluor 488 keçi anti-tavşan IgG (H + L) </td><td> Invitrogen / Moleküler Probları </td><td> A-11034 </td><td></td></tr><tr><td> Alexa Fluor 488 keçi anti-fare IgG (H + L) </td><td> Invitrogen / Moleküler Probları </td><td> A-11.029 </td><td></td></tr><tr><td> 4&#39;6-diamidino-2-fenil-indol dihidroklorür (DAPI) </td><td> Invitrogen / Moleküler Probları </td><td> D1306 </td><td></td></tr><tr><td> Altın anti-fade montaj orta uzatmak </td><td> Invitrogen / Moleküler Probları </td><td> P36930 </td><td> Kullanmadan önce oda sıcaklığına </td></tr><tr><td> Fisherfinest Premium Kapak Cam </td><td> Balıkçı </td><td> 12-544-14 </td><td></td></tr><tr><td> Floresan mikroskop </td><td> Zeiss </td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

Bakteriyel yüzey proteinleri, konak hücreleri ile doğrudan temas halinde ve 1 çevre besin alımını ilgilidirler . Bu nedenle, hücresel yerleşimi bakteriyel proteinlerin fonksiyonel bir rol içgörüler sağlayabilir. Bakteri proteinleri Yüzey yerelleştirme patojenite mekanizmaları içinde yer virülans faktörleri belirlenmesi yolunda önemli bir adım. Leptospiral membranlar 2-5 fraksiyonasyon Yöntemleri lipoproteinler transmembran OMPs vs gibi dış membran proteinleri (OMPs), belirli bir sınıf için seçici olması ve bu nedenle ması yol açabilir . Bu büyük olasılıkla yapısal farklılıklar ve dış membran ile ilişkili nedeniyle. Lipoproteinler, N-terminal lipid benzer parçaları (üç yağ asitleri) ve lipid bilayeri fosfolipit 6, 7 arasında bir hidrofobik etkileşim yoluyla membran ile ilişkilidir . Buna karşılık, transmembran OMPs genellikle β-yaprak amphipathic beşik gibi yapı 8, ​​9 düzenlenen çift katlı lipid içine entegre edilmiştir. Buna ek olarak, dış membran protein varlığı, protein ya da kendi etki alanı yüzeyinde maruz kaldığı garanti değildir. Spirochetal dış membranlar, alt yüzey proteini hücre ve içerme nazik manipülasyon içeren kırılgan ve bu nedenle gerektiren yöntemler olarak bilinen dış membran bütünlüğünü değerlendirmek için kontrol eder. Burada, doğrudan sağlam leptospires proteinlerin yüzey maruziyeti değerlendirmek için immünofloresan assay (IFA) yöntemi mevcut. Bu yöntem, antijen-spesifik antikorların leptospiral yüzey proteinleri tanıma dayanır. Burada, 10 OmpL54 için spesifik antikorların yerli, yüzey maruz epitoplar aftero bağlayıcı detetcted. Sağlam karşı permeabilize hücrelerin antikor reaktivitesi karşılaştırılması bir protein seçici leptospiral yüzeyinde mevcut olup olmadığını ya da hücresel dağılımı değerlendirilmesini sağlar. Dış membran bütünlüğünü tercihen periplasm bulunan bir veya daha fazla yeraltı protein, antikor kullanarak bir şekilde değerlendirilmelidir. Yüzey IFA yöntemi spesifik antikorların mevcuttur leptospiral protein yüzey maruz analiz etmek için kullanılabilir. Yöntemin kullanışlılığı ve sınırlama istihdam antikorlar yerli epitoplar bağlamak mümkün olup olmadığını bağlıdır. Rekombinant proteinler, yerli, yüzey maruz proteinlerin epitoplar karşı antikorları sıklıkla gündeme olduğundan kabul edilmeyecektir. Bununla birlikte, yüzey IFA yöntemi sağlam bakteriyel yüzeyler bileşenleri çalışmak için değerli bir araçtır. Bu yöntem sadece leptospires değil, aynı zamanda diğer spiroket ve gram-negatif bakteriler için değil uygulanabilir. OMPs yüzey pozlama ile ilgili güçlü sonuçlar için, birçok hücre lokalizasyon yöntemleri içeren geniş kapsamlı bir yaklaşım 10 tavsiye edilir .

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Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Leptospiral proteinlerin yüzey pozlama değerlendirmek için etkili bir yöntem açıklanmıştır. Bu yöntem özellikle kırılgan leptospiral hücrelerin dış membran kesintileri önlemek için tasarlanmıştır. Bu teknik, dış membran ve antikor reaksiyonu özgüllüğü bütünlüğünü değerlendirmek için birkaç negatif kontroller istihdam gerektirir.

Yüzey maruz Leptospiral Proteinler immuno-floresans Testi

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

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JoVE Journal

Immunology and Infection

15.7K Görüntüleme.  University of California, Los Angeles. Leptospiral proteinlerin yüzey pozlama değerlendirmek için etkili bir yöntem açıklanmıştır. Bu yöntem özellikle kırılgan leptospiral hücrelerin dış membran kesintileri önlemek için tasarlanmıştır. Bu teknik, dış membran ve antikor reaksiyonu özgüllüğü bütünlüğünü değerlendirmek için birkaç negatif kontroller istihdam gerektirir.

Video: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Gerçek zamanlı, TCR Eşzamanlı Görüntüleme ve İlişkili Sinyal Proteinler için TIRF Mikroskopi Tekniği

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

İmmünoloji ve Enfeksiyon

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Mürin Norovirüs için Plak Testi

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Floresan Etiketli Vaksinia Virüs Proteinlerinin Verimli Üretimi için Metodoloji

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Faj Phenomics: Fizyolojik Yaklaşımlar Roman Viral Proteinler karakterize etmek

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenavirüs hücre Ek Ölçümü için son derece hassas Deneyi

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Bir Kantitatif Mikro-nötralizasyon Testi optimizasyonu

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Proteinlerin içinde Planta serilerinde Plasmodesmal yerelleştirme tanımlaması

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Sistein Thiols rekombinant proteinler Vitro siteye özgü Glikozilasyon için hedefleme

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.