Biz yazının eleştirel okuma için Dr Vesper Fe Marie Ramos teşekkür etmek istiyorum. Bu çalışma Araştırma Kaynakları Ulusal Merkezi (P20 RR0115635-6, C06 RR17417-01 ve G20RR024001) ve Ulusal Nörolojik Bozukluklar Enstitüsü ve İnme (2R01 NS052609) tarafından desteklenmiştir.

1. Ekipman hazırlanması <ol><li> Bir Misonix 3000 veya 37 sabit bir sıcaklıkta tutmak için bir Thermo Electron NESLAB EX-7 su banyosu (Newington, NH) bağlı Misonix 4000 sonikatör (Farmingdale, NY) ° C kullanın Thermo Scientific (Waltham, MA) dan elde edilen kubbeli kapak ile 200 ul ince duvarlı tüp PCR şeritler örnekleri sonikasyon. </li><li> Yepyeni sonikatör sürekli çalışma 9 bir &quot;soygun&quot; dönemi gerektirir. Bir iki ay aradan-in dönemi 10 ayarlanmış genlik seviyesi ile 40 saniye sonication patlama ve 10 dakikalık inkübasyon döngüleri oluşur. Bir titanyum mikroplaka boynuz (Şekil 1, panel B) yüzeyi boyunca erozyon uyulması gerekir. Dolaşan su titanyum mikroplak boynuz erozyon nedeniyle beyaz bulanık görünecektir. Günlük bu suyu değiştirin. </li><li> PrP Sc amplifikasyon mikroplak boynuz boyunca değişir. En iyi PrP Sc amplifikasyon verim içinde elde edilebilirmikroplak boynuz (Şekil 1, panel C&#39;ye bakınız) merkezine 5 cm yarıçapında. </li><li> PMCA biri yuvarlak 144 döngüleri oluşur. Her döngü 5 saniye ile 10 dakika sonikasyon inkübasyondan oluşur. Bu kabaca PMCA yuvarlak başına 24 hr gerektirecektir. </li><li> Sonikatör kontrol panelinde görüntülenen sonication ve güç çıkışı, sonikatör en raf ve su banyosundan dolaşan su sıcaklığı mevcut PCR tüpleri sayısına göre değişir. Su 37 ° C&#39;de dengelenmiş olduğundan emin olun, ve sonikatör en tüp raf (Şekil 1, panel C)% 30&#39;dan fazla doldurmayın. Mikroplak boynuzu ile PCR tüpleri duyurun. PCR tüp stripleri arasındaki boş alan en az bir satır var ve (Şekil 1, panel C) şerit başına birbirine bağlı en fazla dört tüpler sağlar. </li><li> Sonikasyon gücü başarılı bir prion suşu amplifikasyonu için kritik önem taşımaktadır. Sonicating iken, bir güç çıkışı için genlik ayarlamak155 ~ 170 watt arasında değişen. Laboratuvarımızda güç seviyesi sırasıyla altı ve Misonix 3000 ve Misonix 4000 için yedi, ayarlanır. </li><li> Sert su lekeleri birikmesini önlemek için su banyosunda distile su kullanın. Bu su haftalık değiştirin. Kuluçka ve sonication çevrimleri sırasında, su yoğuşması mikroplak korna kapağı içinde meydana gelecek. Şekil 1, panel A&#39;da gösterildiği gibi yoğunlaşmayı önlemek için, sonikatör muhafazası kutusunun üstüne küçük bir akvaryumda ısı pedi takın </li></ol> 2. Hazırlanıyor <ol><li> Hayvanların kullanıldığı bütün işlemler Creighton Üniversitesi Kurumsal Hayvan Bakım tarafından onaylanan ve Kullanım Kurulu ve Laboratuar Hayvanlarının Bakımı ve Kullanımı Kılavuzu ile uyumlu yapılmıştır. Herhangi bir doku toplama önce, hayvan anestetize ve transkardiyal buz gibi soğuk fosfat ile perfüze olmalıdır beyin kan kaldırmak ve inhibisyonu önlemek için pH 7.4, 5 mM EDTA ile tamponlanmış tuz çözeltisiKan 7 tarafından PMCA reaksiyon. Aseptik teknik kullanarak hızla hayvan dokuları toplayın ve her prion suşu için özel diseksiyon araçları vardır. Diseksiyonu sonra, 1.5 ml mikrosantrifüj tüpünün içindeki doku yerleştirmek, flaş kuru buz üzerinde dondurma ve -80 saklayın ° C homojenizasyon kadar. </li><li> PMCA alt tabaka (PrP C) çözelti hazırlamak için, bir Tenbroeck doku öğütücü kullanılarak buz gibi soğuk tampon dönüştürme ile bir% 10 ağırlık / hacim (ağırlık / hacim) çözeltisi (parça 3) için bir etkilenmemiş beyin hamsteri (BM BH) homojen hale getirilir. Homojenleştirici iken, çözüm köpük aşırı önlemek için taşlama içine hava almak için dikkatli olun. 30 sn ve kısım 1.5 ml mikrosantrifüj tüplerine süpernatant için 500 xg&#39;de santrifüj çözümü netleştirin. -80 ° C&#39;de saklayın, daha sonra kullanmak kadar. </li><li> PMCA tohum (PrP Sc) çözeltisi hazırlamak için, buz Wate ile% 10 w / v çözüm bulaşıcı bir malzeme (beyin, dalak, lenf düğümleri, diğer dokulara veya kontamine toprak) homojenize0,6 ml mikrosantrifüj tüpleri r ve kısım çözüm. -80 ° C&#39;de saklayın, daha sonra kullanmak kadar. </li></ol> 3. Dönüşüm Tampon Hazırlanması <ol><li> 50 ml&#39;lik bir balon jojeye (sonuç Triton X-100,% 1 son konsantrasyon) (; Steinheim, Almanya Sigma-Aldrich GmbH) içinde Triton X-100, 0,5093 g tartılır. </li><li> Biri Komple Proteaz İnhibitör Tablet (; Mannheim, Almanya Roche Diagnostics GmbH) ekleyin. </li><li> EDTA 0,0931 g (bu EDTA, 5 mM nihai konsantrasyon neden olur (), Phillipsburg, NJ Mallinckrodt Baker, Inc) ekleyin. </li><li> 1N NaOH ile pH 7.4 &#39;e ayarlayın. </li><li> Dulbecco Fosfat ile 50 ml hacim ayarlama (; Manassas, VA Mediatech Inc) (DPBS) Tamponlu Tuzlu. </li><li> Bir vakumlu filtrasyon cihazı ile bir 0.45 mikron nitroselüloz membran ile çözelti filtre. Dönüşüm tampon fazla bir ay için 4 ° C sıcaklıkta saklanabilir. </li></ol> 4. PMCA kullanma Prion Suşlarının Amplifikasyon <ol><li>PCR tüp içine 1:20 oranında (PMCA tohum ve PMCA alt tabaka 95 ul, yani karışım 5 ul) de PMCA alt tabaka içine PMCA tohum seyreltilir. -80 15 ul kısım çıkarın ve saklayın ° unsonicated kontrolü için C; Bu unsonicated kontrolü Western Blot (WB) analizi takip proteinaz K (PK) sindirim yoluyla PrP Sc amplifikasyon ölçmek için kullanılacaktır. İlave bir kontrol unsonicated PMCA reaksiyon süresi boyunca 37 ° C sıcaklıkta bir ikinci 15 ul alikot yerleştirilmesi ile oluşturulur olabilir. Üç tekrarları en az istatistiki amaçlar için gereklidir. </li><li> Konu Şekil 1 ile ilgili olarak tarif PMCA bir tur için örnek geri kalanı (70 ul) eklendi Panel A. çalkalayın tüpler PCR tüpün kubbeli kapak üzerinde su yoğuşmayı önlemek için her 5 saat. </li><li> Numune tüpler aşağı döndü ve yavaşça homog sağlamak için herhangi bir işlem sonrası açmadan önce (çözüm kubbeli kapağı girmeyiz dikkatli olmak) vortekslenerek edilmelidireneity ve potansiyel kontaminasyon. </li><li> Kısa bir kuluçka süresi suşları (örneğin, HY TME HaCWD, ya da 263K) seri PMCA (sPMCA) için taze enfekte olmamış 95 ul içine yuvarlak bir ikinci sonicated örnek 5 ul transfer ederek 1:20 oranında sonicated madde seyreltik beyin homojenatı (Şekil 2, Panel B). </li><li> Uzun kuluçka süresi suşları (örneğin, DY TME, 139H, 22CH, 22AH, ya da ME7H) arasında sPMCA için, enfekte olmamış taze 50 ul içine yuvarlak bir ikinci sonicated örnek 50 ul transfer ederek 1:1 oranında sonicated madde seyreltik beyin homojenatı (Şekil 2, Panel B). </li><li> Önceden seyreltilmiş sonicated karışımı (adımlar 4,4 ya da 4,5) iki alikotları 15 ul çıkarın ve -80 az biri depolamak 37 ° C&#39;de ve diğer ° C (PMCA boyunca iki unsonicated kontrol çözümleri). Bir PMCA yuvarlak iki kalıntıları koşuluyla. </li><li> Bu prosedür, süresiz olarak tekrar edilebilir. Laboratuvarımızda, bizdeğişmeden kalan zorlanma özellikleri ile, onbeş PMCA mermi kadar gerçekleştirdik. </li><li> PK örnekleri sindirimi. Tipik bir DB, 5 örnek PK 80 ug / ml ila 5 ul (bu çözelti 40 ug / ml &#39;lik bir nihai konsantrasyon PK sahip olacaktır) ul ve bir saat boyunca 37 ° C&#39;de inkübe edilir karıştırın. Jel yükleme tamponu 10 ul ekle. 10 dakika için bu çözelti kaynatılır. Çözüm soğumasını ve jel içine 10 ul yük olsun. </li><li> Amplifikasyon başarı hesaplamak için ya bunları karşılaştırarak amplifiye PrP Sc miktarını tahmin etmek için PK-sindirilmiş örnekler üzerinde WB analizi gerçekleştirmek, unsonicated kontrolleri 20,21 Şekil 2, Panel C veya bilinen miktarda rekombinant PK-sindirilmemiş PrP. </li></ol> 5. Çapraz bulaşma kaçınmak Kritik adımlar çapraz kontaminasyon ve proteaz dayanıklı ürünler de novo oluşumu nedeniyle yanlış pozitif oluşumunu en aza indirmek için alınması gerekir. </p&gt; <ol><li> Her prion suşu için numune toplama için diseksiyon araçları özel bir kümesi (yani forseps, cımbız, makas) kullanın. </li><li> PMCA bir alt tabaka olarak kullanılmak üzere etkilenmemiş beyin homojenleştirme öncesinde,% 1 çamaşır suyu ile pipetler temizleyin. 10 dakika süreyle% 1 çamaşır suyu ile sonikatör en PCR tüp raf, doku homojenizatör ve PCR tüp depolama rafları ıslatın ve distile su ile iyice durulayın. </li><li> Bir taze Versi-kuru Lab Soaker yeni bir numune homojenize edilmesi için her zaman birlikte çalışma alanı Kapak, bir yeni deneme PMCA hazırlanması için, ya da sPMCA mermi arasında aliquoting. </li><li> Yeni eldivenler yeni örnekler (sPMCA mermi arasında alikotları aktarırken özellikle) ele alınır, her zaman kullanın. </li><li> Her PMCA döngü için kısa sonication patlamaları kullanın. 10 dakika inkübasyon ile birlikte 5 saniye sonication döngüleri de novo PrP Sc 1,20,21,23,24 oluşturmadan prionlar yükseltmek için yeterlidir. Diğer laboratuarlar gösterildiği gibi, artansonikasyon zaman, sonication genlik artırmak ve PMCA döngü sayısı uzanan de novo PrP Sc oluşumu 2 için olasılığını arttırmaktadır. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

Yükseltici bulaşıcı prion proteinlerin Zorluklar in vivo deneyler uzun inkübasyon süreleri ve giderleri vardır. PMCA tekniği bulaşıcı prion ajanları güçlendirmek için uygun maliyetli bir yoludur. Birkaç laboratuar doğru vitro 7, 9, 12, 14, 19,24 içinde prion suşları yükseltmek için PMCA yeteneğini doğruladı. Prion hastalıkları türler arasında aktarılabilir. Bessen ve Marsh etkili iki ayrı hamsterden türetilmiş prion suşl...

<table border="1"><tbody><tr><td> Reaktif / Ekipman </td><td> Üretici </td><td> Kedi. Numara </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck Doku Öğütücü </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> NESLAB EX-7 Su Banyosu </td><td> Thermo Electron </td><td> NESLAB EX-7 </td></tr><tr><td> 0.2 ml PCR Tüp Şeritler </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Komple Proteaz İnhibitör </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Versi-Kuru Lab soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Isıtıcı </td><td> Zoo Med Laboratories, Inc </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Prionlar hayvan ve insanlarda 9,18 olarak kaçınılmaz ölümcül bulaşıcı süngerimsi ensefalopati (TSE) neden bulaşıcı ajanlardır. Prion proteini iki farklı izoformu, bulaşıcı olmayan ana-kodlanmış protein (PrP C) ve enfeksiyöz protein (PrP Sc), PrP C 8 anormal katlanmış izoformu vardır. Prion ajanlar ile çalışan zorluklardan biri öncesinde ev sahibi inokülasyon 13 sonrasında klinik bulguların gelişimi uzun bir kuluçka dönemidir. Bu geleneksel olarak uzun ve pahalı hayvan biyoassay çalışmaları görevlendirdi. Ayrıca, PrP Sc biyokimyasal ve biyofiziksel özelliklere kötü onların sıradışı konformasyon ve agregasyonu devletler nedeniyle karakterize edilir. PrP Sc in vitro 14 PrP Sc PrP C tohum dönüşüm yapabilirsiniz. PMCA Adva alır bir in vitro bir tekniktirPrP C ve PrP Sc tohum 19 dakika miktarları aşan miktarda içeren bir sistem, giderek artan bir oranda, PrP Sc büyük miktarlarda üretmek için sonication ve inkübasyon döngüleri kullanarak bu yeteneği ntage. Bu teknik, etkili prion zorlanma girişime taklit etmek, PrP C PrP Sc dönüştürme türleri ve gerilme özgüllük özetlemek için, ve enfekte dokuları, sıvıları ve çevresel örneklerde 6,7,16 gelen PrP Sc çok düşük seviyelere yükseltmek için kanıtlanmıştır 23. , Kontaminasyonu en aza tutarlı sonuçlar üreten, ve bu sonuçların ölçülmesi için öneriler içeren bu kağıdı ayrıntıları PMCA protokolü. Biz de bulaşıcı prion suşları, prion zorlanma girişim, ve çevre Prionların algılama üretimi ve karakterizasyonu gibi çeşitli PMCA uygulamaları tartışmak.

Protein misfolding siklik amplifikasyon (PMCA) İn vitro 7 PrP, Sc, 12, 14, 19, 24, yükseltmek için kullanılır. Başarılı amplifikasyon PrP Sc, Şekil 3&#39;te gösterildiği gibi PK-dirençli prion proteini (hamsterden türetilmiş prion suşları için 19 ila 30 kDa geçiş) Western blot üzerinde bant yoğunluğunda bir artış ile gösterilmiştir. Kendi bant yoğunluk artışı sonra PMCA PK-dirençli PrP Sc malzeme amplifika...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Protein misfolding siklik amplifikasyon (PMCA) Prion dönüşüm ve zorlanma ve tür engellerin çalışma için bir in vitro testtir. Aynı zamanda, bir prion saptama sistemi olarak da kullanılabilir.

Prionlar protein misfolding Döngüsel Amplifikasyon

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K Görüntüleme.  University of Nebraska at Lincoln. Protein misfolding siklik amplifikasyon (PMCA) Prion dönüşüm ve zorlanma ve tür engellerin çalışma için bir in vitro testtir. Aynı zamanda, bir prion saptama sistemi olarak da kullanılabilir.

Video: Protein Misfolding Cyclic Amplification of Prions

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

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

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

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

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

İmmünoloji ve Enfeksiyon

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

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

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

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Türler özgü Algılama Loop-aracılı İzotermal Amplifikasyon (LAMP) Tahliller<em&gt; Eimeria</em&gt; Tavuklar bulaştırmak ki

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

tespiti için Liyofilize Döngü aracılı İzotermal Amplifikasyon Reaktifler Gelişimi<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

Gerçek zamanlı depremin dönüşüm tahlil CWD Prionlar fekal madde olarak algılanması için kaynaklı

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Bimolecular tamamlayıcı benzeşme arıtma (BiCAP) tarafından parçalanmış çok protein sinyal kompleksleri

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Otomatik davranış analizi büyük C. elegans nüfus kullanarak bir geniş alan bakış açısı izleme platformu

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Üretimi, güçlendirme ve titrasyon rekombinant solunum sinsisyal virüs

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Tilapia Lake Virüs Ünün Özel ve Hızlı Tespiti İçin Ters Transkripsiyon Döngü Aracılı İzomal Amplifikasyon (RT-LAMP) Testi

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.