1. Nicotiana benthamiana Bitkiler Virüs Aşılaması ve VLP&#39;ler Arıtma <ol><li> Patates X virüsü (PVX) tabanlı taşıyan vektör plasmidlerden şapkalı T7 RNA transkriptleri üretin MRFV wild-tip (wt) ve Cys-mutasyona uğramış kılıf protein (CP) genleri 12, Ambion en T7-mMessage mMachine Kit kullanarak. </li><li> Her T7 transkript reaksiyon için, N., iki tam genişletilmiş yaprakları aşılamak 10 ul reaksiyon ile ve 25 ışık (25,000-30,000 lux) ile 16 saat süreyle% 60 nem, serada 10 gün için inkübe edilir benthamiana bitkiler 20 ° C&#39;de ve 8 saat karanlık ° C. </li><li> Harves t N. benthamiana virüs bulaşmış yaprakları 10 gün sonrası aşılama (dpi), buz üzerinde bitki doku (50 gram) ve yer tartın. </li><li> 0.5 M Na-sitrat tampon, pH = 5.5 (kullanımı 2 bitki gram doku başına tampon ml) içinde soğutulmuş bir çözeltisi, 100 ml varlığında, bir karıştırıcı içinde bitki materyali homojenize edilir. </li><li> Cheesecl 3 kat boyunca Homojenat Filtreoth ve 30 dakika boyunca 27,000 x g&#39;de santrifüj ile netleştirmek. Soğutulmuş mezun silindirin içine supernatant aktarın, hacmini ölçmek ve sonra soğutulmuş steril behere aktarılır. Pelet atın. </li><li> 4 ° C sıcaklıkta 15 dakika için karıştırılır,% 10 polietilen glikol 8000 (PEG 8000) ilave edilerek süpernatant işleme ve 4 de ilave bir 45 dakika süreyle inkübe ° C virüs benzeri tanecikler (VLP&#39;ler)-PEG matris çökeltilir. </li><li> 4 ° C&#39;de 20 dakika boyunca 27,000 x g numune santrifüjlenir ve süpernatant çıkarın. </li><li> 0.05 M Na-sitrat tamponu, 5.5 2 içeren% Triton X-100, pH 8 ml ilave edilerek pellet işlemden geçirin. Swirl santrifüj tüplerine de pelet yayınlayacak. Soğutulmuş steril behere pelet ve tampon aktarın. Aynı tampon, 2 ml ile hemen tüpler yıkayın ve behere ekleyin. Pelet (genellikle 1 saat) çözülene ° C kadar 4 de süspansiyon karıştırın. </li><li> 27,000 x g&#39;de 5 dakika için santrifüjleme ile netleştirmek için pelet atmak ve p,4 de 2 saat boyunca 140,000 x g&#39;de santrifüj ile roses süpernatant ° C. </li><li> 1 ml 0.05 M Na-sitrat tampon, pH 5.5 &#39;de pelet yeniden süspanse edin ve 0.05 M Na-sitrat tampon, pH = 5.5 içinde yapılmış bir% 10-40 oranında sakaroz eğim üzerine süspansiyon yerleştirin. </li><li> 4 de 140,000 xg&#39;de 3 saat boyunca gradyanı Santrifüj ° C. </li><li> Santrifüj tüpünün üst üzerine hafif bir parlaklık ve tüpün üst kısmından 4 cm ışık saçılım üst bant toplamak, 4 az 140,000 xg&#39;de 3 saat için 0.05 M Na-sitrat tamponu, pH 5.5, ve santrifüj ile sulandırmak ° C. </li><li> Altı ay için 4 ° C de steril su deposu ve 0.5 ml içinde pellet yeniden süspanse edin. </li><li> Bir Bradford protein tayini yapılarak VLP&#39;ler konsantrasyonunu ölçmek. VLP&#39;ler verimi bitki dokusunun 1 ila 3 mg / g&#39;dır. </li></ol> 2. VLP&#39;ler arasında Flöresein-5-maleimid-etiketleme Reaksiyonları <ol><li> 50 mM sodyum fosfat içinde fluoresein-5-maleimit (427,37 g / mol) bir 1 mM çözelti hazırlayınE tamponu, pH 7.0, 1 mM EDTA. Vorteks kullanılarak karıştırılır ve tam çözünme doğrulayın. Alüminyum folyo ile ışıktan tüp koruyun. </li><li> Adım 1 ve karıştırın üretilen VLP&#39;ler fluoreseinin-5-maleimid ekleyin. Sülfhidrilleri molar miktarda fluoresein-5-maleimit bir 25 kat molar fazlalık kullanın. Genel olarak, 1 ug / ml &#39;den ve fluoresein-5-maleimit solüsyonu 60 ul arasında derişimde VLP&#39;ler 30 ul kullanılarak kabul edilebilir sonuçlar elde edilmektedir. </li><li> 4 ° C&#39;de ya da oda sıcaklığında gece boyunca 2 saat boyunca reaksiyon inkübe </li><li> 50 mM bir son konsantrasyon için ditiyotretol (DTT) ilave edilerek tepkime sona erdirme. </li><li> İzleme olmayan reaksiyona oda sıcaklığında 1,500 x g&#39;de 2 dakika süre ile bir tuzsuzlaştırma spin kolon (Thermo Scientific Inc, Rockford, IL) ve santrifüj kullanılarak floresein. </li><li> SDS-PAGE analizi için, her bir reaksiyon 10 ul 2 x SDS-PAGE Laemmli numune tamponu 10 ul ilave edin. Yukarı bir ay ya da için 4 ° C&#39;de ışıktan koruyarak etiketli protein saklayın3 aya kadar -20 ° C&#39;de tek kullanımlık alikotları. </li></ol> 3. Biotin Kuruluş ve reaksiyon Belirlenmesi Etiketleme <ol><li> Su, fosfat tamponlu tuzlu su (PBS), 1. Adımda üretilen VLP&#39;ler arasında pH değeri 7.0 olan bir tampon alışverişi yapmak için tuzsuzlaştırma sıkma kolon (Thermo Scientific Inc, Rockford, IL) kullanın. </li><li> Aşağı yukarı pipetleme ve tarafından maleimid-PEG2-biotin ve mix No-tartılır PBS 190 ul ekleyerek maleimid-PEG2-biotin bir 20 mM stok solüsyonu hazırlayın. </li><li> VLP&#39;ler ve karıştırın maleimid-PEG2-biotin ekleyin. VLP&#39;ler çözümler için bir ayıraç ile 10-kat molar fazlalığı kullanmak ≤ 2 mg / ml idi. </li><li> 4 saat için oda sıcaklığında reaksiyon inkübe edin. </li><li> İzleme olmayan reaksiyona oda sıcaklığında 1,500 x g&#39;de 2 dakika süre ile bir tuz giderme spin kolon (Thermo Scientific Inc, Rockford, IL) ve santrifüj kullanılarak maleimid-PEG2-Biotin. </li><li> Pierce Biotin kantitasyonu Takımı (Thermo Scientific) ve fo kullanarak VLP&#39;ler mol başına biotin mol belirleyinüreticinin talimatlarına llowing. </li></ol> 4. Cys 2-VLP&#39;ler ve F Peptid Çapraz bağlama <ol><li> Adım 2. (2-Cys VLP&#39;ler) çapraz bağlama için mevcut sistein kalıntıları ile sonuçlanmıştır VLP&#39;ler aşağıdaki adımda kullanılır. Dimetil sülfoksit 680 ul NHS-PEG4-maleimid çapraz bağlayıcı 10 mg çözülerek taze bir çapraz-bağlayıcı stok çözeltisi (28.63 mM) hazırlayın. </li><li> , 17 (F) çözülür-amino konjugasyon tamponu 50 ul (1 x PBS, pH 7.2, 1 mM EDTA) içinde asit uzunluğunda peptid (LLPNMOKDKEACAKAPL) 0.1 mM bir konsantrasyonda. </li><li> 1 mM nihai konsantrasyonunda (ki peptid solüsyonu, 0.1 mM için bir 10-kat molar fazlası tercih edilir) çözündürüldü peptit (protein-NH2) 50 ul çapraz bağlayıcının 4 ul ekle. </li><li> 4 ° C&#39;de 2 saat inkübe reaksiyon </li><li> 1,500 x g&#39;de santrifüj ile, konjugasyon tampon (1 mM EDTA 1 x PBS, pH 7.2) ile dengelenmiş bir tuz giderme kolonu (Thermo Scientific Inc, Rockford, IL) kullanılarak çapraz bağlı peptit arındırmakOda sıcaklığında 1 dakika için. </li><li> VLP&#39;ler (protein-SH) ve reaksiyon ile VLP&#39;ler ile peptid arasında bir moleküler ağırlık arasındaki fark olarak dahil tioller ve aktive edilmiş aminlerin sayısına göre belirlenen bir molar oranda çapraz bağlı peptit (protein-NH2) birleştiren bir reaksiyon karışımı ayarlayın. </li><li> 4 ° C&#39;de 2 saat için oda sıcaklığında reaksiyon karışımı inkübe </li><li> Western blot analizi için, Laemmli tamponu, 10 dk için kaynatılır, ile elde edilen konjüge ürün 1:1 Karışım ve% 10-20 Tris-Glisin jeli (Invitrogen) ve bir ön-dökme üzerinde SDS-PAGE ile devam edin, bir üzerine electroblotting takiben Nitroselüloz zar (Invitrogen). 1 x PBS, pH 7.2% 0.05 Tween 20 (PBS-Tween) ve uygun fosfataz-etiketli ikincil antikor tarafından takip peptit-spesifik antikorlar ile% 5 kaymağı alınmış süt tozu ve sonda içeren membranlar ile engelleme. </li></ol>

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Yayında ticari ürünlerin ticaret ünvanlı Mansiyon spesifik bilgilerin sağlanması amacıyla sadece ve ABD Tarım Bakanlığı tarafından tavsiye veya onaylandığı anlamına gelmez.

Burada sunulan yöntem, bitki üretilen VLP&#39;ler yüzeyi üzerinde hem de diğer protein kompleksleri üzerinde mevcut reaktif sistein çok hassas ve hızlı bir analizine izin verir. Maleimides stabil tiyoeter bağları oluşturmak için ücretsiz sülfidril içeren moleküllerle reaksiyona tiyol özgü reaktifler vardır. Bu yöntem, diğer amino asitler ile etkileşimlerine sülfhidril grupları ile tepkimeye maleimides özgüllüğünü dayanmaktadır. VLP&#39;ler arasında doğal yapısının korunması tüm sü...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Thinwall, Ultra-Clear Tüpler </td><td> Beckman </td><td> 344059 </td><td></td></tr><tr><td> mMESSAGE mMACHINE T7 Takımı </td><td> Yaşam Teknolojileri </td><td> AM1344M </td><td></td></tr><tr><td> Fluoresein-5-Maleimid </td><td> Thermo Scientific Life Teknolojileri </td><td> 46130 F150 </td><td> 46130 F150 ile sipariş yerine dışında </td></tr><tr><td> Pierce Biotin Kantifikasyonu Takımı </td><td> Thermo Scientific </td><td> 28005 </td><td></td></tr><tr><td> EZ-Link Maleimid-PEG2-Biotin, Biçim No-Weigh </td><td> Thermo Scientific </td><td> 21901 </td><td></td></tr><tr><td> SM (PEG) n Çapraz Bağlayıcılar </td><td> Thermo Scientific </td><td> 22107 </td><td></td></tr><tr><td> % 10-20 Tris-Glisin jeli </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Laemmli Tampon </td><td> Bio-Rad </td><td> 1610737 </td><td></td></tr><tr><td> Tris Glisin SDS Buffer Koşu </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> Tris Glisin Transferi Tampon </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> Nitroselüloz Membran Filtre Kağıdı Sandviç </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> Tavşan IgG antikor Arıtılmış Fosfataz Etiketli Affinity </td><td> Kirkegaard ve Perry Laboratuvarları </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP Fosfataz Yüzey </td><td> Kirkegaard ve Perry Laboratuvarları </td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

Virüs özellikleri ve fiziko-kimyasal ve fiziksel özelliklerini taklit ve sömürücü dünyanın en acil sorunlarından bazılarına çözümler sunmak için umut vaat ediyor. Sırf aralığı ve ilginç özellikleri ile birleştiğinde virüs tipleri potansiyel virüs tabanlı teknolojilerde uygulamalar için sonsuz fırsatlar verecektir. Virüsler, sıcaklık ve pH koşulları altında geniş bir yelpazede farklı şekil ve boyut, simetri özgüllüğü, polyvalence, ve sabit özelliklere sahip partiküller halinde kendini monte etmek özelliğine sahiptir. Doğal olarak, bu gibi özelliklerinin dikkate değer bir dizi ile, virüs biomateryaller 9, aşılar, 14, 15, elektronik maddeler, kimyasal araçlar, konteynırlar ve moleküler elektronik 4, 5, 10, 11, 16, 18, ​​12 olarak kullanım için önerilmiştir. Nanoteknolojide virüsler kullanılması için, yeni işlevler vermek için kendi doğal formlardan değiştirilmesi gerekir. Bu zorlu process gen viral genomun değişiklik ve kimyasal olarak virüs parçacığı reaktif gruplar 8 ile yabancı ya da arzu edilen moleküllerin bağlanması dahil çeşitli mekanizmalar vasıtasıyla gerçekleştirilebilir. Bir virüs değiştirme yeteneği öncelikle virüs fizikokimyasal ve fiziksel özelliklerine bağlıdır. Buna ek olarak, genetik veya fizikokimyasal olumsuz değişiklikler virüsün doğal yapısını ve fonksiyonunu etkileyen virüs olmadan gerçekleştirilmesi gerekir. Maize rayado fino virüsü (MRFV) kat proteinler, protein-protein stabilize edilmektedir kararlı ve boş VLP&#39;ler üreten E. coli içinde kendi kendine bir araya etkileşimi ve bu virüs bazlı teknolojileri uygulamalar 8 kullanılabilir. Tütün bitkilerinde üretilen peptidlerin VLP&#39;ler çeşitli kovalent 13 görüntülenebilir hangi bir yapı iskelesi olarak incelenmiştir. Burada, bir virüs kapsid içinde çözücü erişilebilir sissteinler hangi modifikasyonu için kullanılabilir belirlemek) 1 adımları açıklarkatyon, ve 2) capsids ile değiştirilmiş peptitler bioconjugate. Doğal ya da mutationally takılan amino asit kalıntıları ve standart bağlama teknolojileri kullanarak, malzemeler çok çeşitli böyle Brom mozaik virüsü 3, karanfil benek virüsü 12, Börülce klorotik benek virüsü 6, Tütün mozaik virüsü gibi bitki yüzeyinde ortaya konulmuştur Virüs 17, Şalgam sarı mozaik virüsü 1 ve MRFV 13.

N. mutant MRFV kılıf protein (CP) genlerin geçici ifadesi bir PVX bazlı vektör üretiminde VLP&#39;ler içinde benthamiana bitki Şekil 1 &#39;de tarif edilmektedir. Modifiye MRFV kılıf protein geni bir PVX tabanlı vektör, pP2C2S 2, (D. Baulcombe, Sainsbury Laboratuvarları, Norwich, İngiltere bir hediye) olarak çoğaltılamaz subgenomik CP organizatörü transkripsiyonel kontrolü altında amplifiye ve sonra yerleştirilir. Yılında In vitro RNA transkr...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Sistein artıkları tiyol grubunun çözücü yönelik analiz etmek için bir yöntem,<em&gt; Mısır rayado fino virüsü</emBir peptid çapraz bağlama reaksiyonu, ardından&gt; (MRFV)-virüs benzeri tanecikler (VLP&#39;ler) tarif edilir. Yöntem, belirli reaksiyonlar için hedef olabilir VLP&#39;ler yüzeyi üzerinde çeşitli kimyasal gruplarından durumu yararlanır.

Sistein Kalıntılarının Solvent Erişilebilirlik Analizi<em&gt; Mısır rayado fino virüsü</emÜretilen&gt; Virüs gibi parçacıklar<em&gt; Nicotiana benthamiana</emVLP&#39;ler için Peptidlerin&gt; Bitkiler ve Çapraz bağlama

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K Görüntüleme.  Agricultural Research Service, United States Department of Agriculture. Sistein artıkları tiyol grubunun çözücü yönelik analiz etmek için bir yöntem, Mısır rayado fino virüsü (MRFV)-virüs benzeri tanecikler (VLP'ler) tarif edilir. Yöntem, belirli reaksiyonlar için hedef olabilir VLP'ler yüzeyi üzerinde çeşitli kimyasal gruplarından durumu yararlanır.

Video: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

&#39;Tek Virüs Genomik&#39; kullanarak Tek viryonlar İzolasyonu ve Genom Analizi

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

İmmünoloji ve Enfeksiyon

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 ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronic minigenomlar İçeren virüs benzeri partiküller ile Biyogüvenlik Seviyesi 2 Koşullarında Ebola virüsü Ömrü Modelleme

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Otoimmünite karşı Kök Hücre türetilmiş antijen-özgü düzenleyici T hücrelerinin gelişimini

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

Yumuşak agar üst tabaka tekniği kullanımı bakteriyel olarak üretilmiş önleyici bileşiklerin taranması için

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

Ham Virion ayıklama DNA analizi yeni virüs genleri keşfetmek için virüslü yaprakları gelen RNA analizi ile birleştirerek

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Viral enfeksiyon ve analiz Drosophila Melanogaster ana bilgisayar virüs etkileşimin kurulması

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

Akış sitometrik ölçüm makrofajlar FcγR Cross-linking yanıt ROS üretim

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...