Nous tenons à remercier le Dr Vesper Fe Marie Ramos pour la lecture critique du manuscrit. Ce travail a été soutenu par le National Center for Research Resources (P20 RR0115635-6, C06-01 et RR17417 G20RR024001) et l&#39;Institut national des troubles neurologiques et des maladies (2R01 NS052609).

1. Préparation de l&#39;équipement <ol><li> Utilisez un 3000 Misonix ou Misonix 4000 sonicateur (Farmingdale, NY) relié à un bain Thermo Electron Neslab EX-7 de l&#39;eau (Newington, NH) pour maintenir une température constante de 37 ° C. Soniquer les échantillons dans 200 pi à parois minces barrettes de tubes PCR avec bouchons bombés obtenus à partir de Thermo Scientific (Waltham, MA). </li><li> Un appareil à ultrasons nouvelle marque nécessite un &quot;break-in&quot; période de fonctionnement continu 9. Une rupture dans le délai de deux mois se compose d&#39;une salve sonication de 40 sec et cycles d&#39;incubation de 10 minutes avec le niveau d&#39;amplitude fixé à 10. On devrait observer l&#39;érosion sur toute la surface de la corne de titane microplaque (figure 1, partie B). La circulation d&#39;eau se penchera trouble blanche due à l&#39;érosion de la corne de titane microplaque. Remplacer cette eau tous les jours. </li><li> L&#39;amplification de la PrP Sc varie tout au long de la corne de microplaques. La meilleure efficacité PrP Sc d&#39;amplification peut être obtenu dansun rayon de 5 cm du centre de la corne de microplaques (voir la figure 1, partie C). </li><li> Un tour de PMCA se compose 144 cycles. Chaque cycle se compose de 5 sonication sec et 10 min d&#39;incubation. Ce sera à peu près entraînent à 24 h par tour de PMCA. </li><li> La puissance du moteur de sonication, affiché sur le panneau de commande de l&#39;appareil à ultrasons, varie avec le nombre de tubes PCR présentes dans la grille de sonication et la température de l&#39;eau circulant dans le bain d&#39;eau. Assurez-vous que l&#39;eau est équilibrée à 37 ° C, et ne remplissent pas plus de 30% du portoir du sonicateur (figure 1, partie C). Répartir les tubes PCR à travers la corne de microplaques. Avoir au moins une rangée d&#39;espace vide entre les barrettes de tubes PCR et de permettre au plus quatre tubes reliés entre eux par bande (figure 1, partie C). </li><li> La force de sonication est essentiel pour une amplification souche de prion succès. Bien sonication, régler l&#39;amplitude d&#39;avoir une puissance de sortiecomprise entre 155 ~ 170 watts. Dans notre laboratoire, le niveau de puissance est réglé sur six et sept pour 3000 et Misonix Misonix 4000, respectivement. </li><li> Utilisez de l&#39;eau distillée dans le bain d&#39;eau pour éviter l&#39;accumulation des taches d&#39;eau dure. Remplacer cette semaine de l&#39;eau. Au cours des cycles d&#39;incubation et sonication, l&#39;eau de condensation se produit à l&#39;intérieur du couvercle corne de microplaques. Pour éviter la condensation, fixez les tampons petit aquarium de la chaleur vers le haut de la boîte de l&#39;enceinte de l&#39;appareil à ultrasons comme le montre la figure 1, le panneau A. </li></ol> 2. Préparation des échantillons <ol><li> Toutes les procédures impliquant des animaux ont été approuvés par les soins de l&#39;Université Creighton animaux institutionnel et l&#39;utilisation comité et se conformer aux Lignes pour le soin et l&#39;utilisation des animaux de laboratoire. Avant tout prélèvement de tissus, l&#39;animal doit être anesthésié et transcardiaque perfusé à l&#39;aide glacée phosphate de solution saline tamponnée avec EDTA 5 mM à pH 7,4 pour enlever le sang du cerveau et d&#39;éviter l&#39;inhibition de laRéaction PMCA par le sang 7. Recueillir les tissus animaux rapidement en utilisant une technique aseptique et des outils de dissection dédiées pour chaque souche de prion. Après dissection, placer le tissu dans un tube de 1,5 ml, flash à geler sur de la glace sèche, et de le stocker à -80 ° C jusqu&#39;à homogénéisation. </li><li> Pour préparer le substrat PMCA (PrP C) solution, homogénéiser un cerveau de hamster infecté (ONU BH) à un 10% en poids / volume (p / v) avec un tampon de conversion glacée (voir partie 3) en utilisant un broyeur de tissu Tenbroeck. Bien homogénéiser, veillez à ne pas prendre l&#39;air à l&#39;intérieur du broyeur pour éviter un moussage excessif de la solution. Clarifier la solution par centrifugation à 500 xg pendant 30 sec et aliquote du surnageant dans des tubes de 1,5 ml microtubes. Conserver à -80 ° C jusqu&#39;à utilisation ultérieure. </li><li> Pour préparer la graine PMCA (PrP Sc) solution, homogénéiser une matière infectieuse (ganglions lymphatiques du cerveau, de la rate, d&#39;autres tissus, ou des sols contaminés) à un 10% p / v de solution avec de la glace froide water et aliquote de la solution dans 0,6 ml microtubes. Conserver à -80 ° C jusqu&#39;à utilisation ultérieure. </li></ol> 3. Préparation du tampon de conversion <ol><li> Peser 0,5093 g de Triton X-100 dans une fiole jaugée de 50 ml (résultat est une concentration finale de 1% de Triton X-100) (Sigma-Aldrich GmbH, Steinheim, Allemagne). </li><li> Ajoutez un inhibiteur de la protéase complète Tablet (Roche Diagnostics GmbH, Mannheim, Allemagne). </li><li> Ajouter 0,0931 g d&#39;EDTA (cela se traduira par une concentration de 5 mM final de l&#39;EDTA) (Mallinckrodt Baker Inc; Phillipsburg, NJ). </li><li> Ajuster le pH à 7,4 avec NaOH 1N. </li><li> Réglez le volume à 50 ml avec de phosphate de Dulbecco saline tamponnée (DPBS) (Mediatech Inc; Manassas, VA). </li><li> Filtrer la solution à travers une membrane de nitrocellulose de 0,45 pm avec un appareil de filtration sous vide. Le tampon de conversion peut être conservé à 4 ° C pendant pas plus d&#39;un mois. </li></ol> 4. L&#39;amplification des souches de prion Utilisation PMCA <ol><li>Diluer la graine PMCA dans le substrat PMCA dans un rapport de 1:20 (mélange soit 5 ul de la graine PMCA et 95 ul du substrat PMCA) dans un tube PCR. Retirez et rangez une aliquote de 15 ul à -80 ° C pour le contrôle unsonicated, ce contrôle unsonicated sera utilisée pour quantifier l&#39;amplification PrP Sc par la protéinase K (PK) digestion suivie par Western blot (WB) analyse. Une commande supplémentaire unsonicated peut être généré en plaçant une seconde aliquote 15 ul à 37 ° C pendant toute la durée de la réaction PMCA. Un minimum de trois répétitions est nécessaire à des fins statistiques. </li><li> Sous réserve du reste de l&#39;échantillon (70 pl) pour une tour de PMCA comme représenté sur la figure 1, partie A. Agiter chaque tube 5 h afin d&#39;éviter la condensation de l&#39;eau sur la coiffe en forme de dôme du tube de PCR. </li><li> Tubes de l&#39;échantillon doit être centrifugé et délicatement vortex (en faisant attention que la solution ne vont pas dans le bouchon en forme de dôme) avant d&#39;ouvrir après toute procédure visant à assurer homogeneity et éviter toute contamination. </li><li> Pour série PMCA (sPMCA) des souches période d&#39;incubation courte (c.-à-HY TME, HaCWD ou 263K), diluer la matière traitée aux ultrasons dans un rapport de 1:20 en transférant 5 pl de l&#39;échantillon soniqué de première ronde dans 95 ul de frais non infecté homogénat de cerveau (figure 2, partie B). </li><li> Pour sPMCA de longues souches période d&#39;incubation (c.-à-DY TME, 139H, 22CH, 22AH, ou ME7H), diluer la matière traitée aux ultrasons dans un rapport de 1:1 en transférant 50 ul de l&#39;échantillon soniqué de première ronde dans 50 pl de frais non infecté homogénat de cerveau (figure 2, partie B). </li><li> Retirez deux 15 aliquotes du mélange préalablement dilué soniqué (des étapes 4.4 ou 4.5) et stocker l&#39;un d&#39;eux à -80 ° C et l&#39;autre à 37 ° C (solutions de contrôle unsonicated pour PMCA deuxième tour). Soumettre les restes d&#39;une tour PMCA deux. </li><li> Cette procédure peut être répétée indéfiniment. Dans notre laboratoire, nousont effectué jusqu&#39;à quinze rounds PMCA, avec les propriétés de déformation reste inchangée. </li><li> PK digérer les échantillons. Pour un type WB, mélanger 5 pl de PK 80 pg / ml à 5 ​​pl de l&#39;échantillon (cette solution a une concentration finale de PK 40 pg / ml) et incuber à 37 ° C pendant une heure. Ajouter 10 ul de tampon de charge de gel. Faire bouillir cette solution pendant 10 min. Laisser la solution refroidir et charger 10 pi dans le gel. </li><li> Pour calculer le succès de l&#39;amplification d&#39;effectuer une analyse sur les échantillons WB PK-digérés pour estimer la quantité de PrP Sc amplifié en les comparant à deux, les contrôles unsonicated 20,21 graphique 2, partie C ou en un montant connu de PK-digérées recombinant PrP. </li></ol> 5. Éviter la contamination croisée Les étapes critiques doivent être prises pour réduire au minimum la contamination croisée et l&#39;apparition de faux positifs dus à la formation de novo de produits résistants à la protéase. </p&gt; <ol><li> Utilisez un ensemble dédié d&#39;outils de dissection (c.-à-pinces, pinces à épiler, ciseaux) pour la collecte de l&#39;échantillon pour chaque souche de prion. </li><li> Avant d&#39;homogénéisation cerveaux sains pour être utilisé comme un substrat PMCA, essuyez les pipettes d&#39;eau de Javel à 1%. Faire tremper le support du tube de PCR sonicateur, homogénéisateurs de tissus, et des supports de stockage de tubes PCR avec l&#39;eau de Javel à 1% pendant 10 minutes et rincer abondamment avec de l&#39;eau distillée. </li><li> Couvrir la zone de travail avec un remous Lab Versi-Dry frais chaque fois qu&#39;un nouvel échantillon doit être homogénéisé, une nouvelle expérience PMCA est d&#39;être prêt, ou lorsque aliquotage entre les rounds sPMCA. </li><li> Utiliser des gants chaque fois de nouvelles de nouveaux échantillons sont traités (en particulier lors du transfert entre les rounds aliquotes sPMCA). </li><li> Utiliser des salves courtes sonication pour chaque cycle de PMCA. Cycles de 5 sonication sec combiné avec 10 minutes d&#39;incubation est suffisant pour amplifier les prions sans générer de novo PrP Sc 1,20,21,23,24. Comme d&#39;autres laboratoires ont montré, en augmentant latemps de sonication, en augmentant l&#39;amplitude de sonication, et s&#39;étendant le nombre de cycles augmente la probabilité PMCA de novo de la PrP Sc formation 2. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

Les défis de amplification protéines prions infectieuses sont les longues périodes d&#39;incubation et les frais de dans des expériences in vivo. La technique PMCA est un moyen rentable pour amplifier les prions infectieux. Plusieurs laboratoires ont confirmé la capacité de PMCA avec précision amplifier souches de prion in vitro 7, 9, 12, 14, 19,24. Les maladies à prions peuvent être transmis entre les espèces. Bessen et Marsh ont effectivement...

<table border="1"><tbody><tr><td> Réactif / équipement </td><td> Fabricant </td><td> Cat. Nombre </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> Grinder tissus Tenbroeck </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Bain Neslab EX-7 Eau </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> 0,2 ml Bandes de tubes PCR </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> Inhibiteur de la protéase complète </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> Soakers Lab Versi-Dry </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Chauffage </td><td> Zoo Med Laboratories, Inc </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Les prions sont des agents infectieux qui causent l&#39;encéphalopathie spongiforme inévitablement fatale transmissible (EST) chez les animaux et les humains 9,18. La protéine prion présente deux isoformes distincts, le non-infectieuse codée par l&#39;hôte de protéine (PrP C) et la protéine infectieuse (PrP Sc), une isoforme anormale pliée de PrP C 8. L&#39;un des défis de travailler avec les prions est la longue période d&#39;incubation avant l&#39;apparition des signes cliniques qui suivent le 13 inoculation d&#39;accueil. Cette traditionnellement chargé longs et coûteux essais biologiques d&#39;origine animale. En outre, les propriétés biochimiques et biophysiques de la PrP Sc sont mal caractérisées en raison de leur conformation inhabituelle et états d&#39;agrégation. PrP Sc peut engendrer la conversion de PrP C en PrP Sc in vitro 14. PMCA est une technique in vitro qui prend avantage de cette capacité en utilisant les cycles de sonication et d&#39;incubation pour produire de grandes quantités de PrP Sc, à une vitesse accélérée, à partir d&#39;un système contenant des quantités excessives de PrP C et des quantités infimes de la graine PrP Sc 19. Cette technique s&#39;est avérée efficace pour résumer la spécificité d&#39;espèce et la souche de PrP Sc conversion de PrP C, à imiter les interférences souche de prion, et d&#39;amplifier de très faibles niveaux de PrP Sc dans les tissus infectés, fluides, et les échantillons environnementaux 6,7,16, 23. Cet article détaille le protocole de PMCA, y compris des recommandations pour minimiser la contamination, des résultats constants, et de quantifier ces résultats. Nous discutons également plusieurs applications PMCA, notamment la production et la caractérisation des souches de prions infectieux, les interférences souche de prion, et la détection des prions dans l&#39;environnement.

Amplification cyclique protéine repliement (PMCA) est utilisée pour amplifier la PrP Sc in vitro 7, 12, 14, 19, 24. Un succès PrP Sc amplification est démontré par une augmentation de l&#39;intensité des bandes sur Western blot de la protéine prion PK-résistante (migration entre 19 et 30 kDa pour souches de prion de hamster dérivés), comme illustré à la figure 3. L&#39;augmentation de l&#39;intensité de la bande après PMCA indique l&#39;amplificati...

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

Protein Misfolding Cyclic Amplification of Prions

Repliement des protéines amplification cyclique (PMCA) est un test in vitro pour l&#39;étude de la conversion du prion et les obstacles souche et l&#39;espèce. Il peut également être utilisé comme un test de détection de prion.

Protein Misfolding Cyclic Amplification des prions

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 vues.  University of Nebraska at Lincoln. Repliement des protéines amplification cyclique (PMCA) est un test in vitro pour l'étude de la conversion du prion et les obstacles souche et l'espèce. Il peut également être utilisé comme un test de détection de prion.

Vidéo: 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.

Procédures d&#39;identification des prions infectieux Après passage dans le système digestif d&#39;une espèce aviaire

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

Recherche

Immunologie et infection

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.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

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.

amplification isotherme (LAMP) Essais de boucle médiation pour la détection des espèces spécifiques de<em&gt; Eimeria</em&gt; Qui infectent les poulets

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.

Le développement de lyophilisé boucle médiées isothermes Amplification Réactifs pour la détection des<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.

En temps réel Quaking induite par le test de Conversion pour détection de CWD Prions dans les matières fécales

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

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Mise en place d’une plateforme de criblage à haut débit afin d’évaluer l’hétérogénéité d’Amplification de gène HER2 dans les Cancers du sein

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

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.

Dissection multi-signalisation Complexes protéiques par Purification d’affinité de complémentation bimoléculaire (BiCAP)

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.

Analyse comportementale des grands c. elegans des Populations à l’aide d’un champ de vision large suivi plate-forme automatisée

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.

Génération, Amplification et titrage du virus Syncytial respiratoires recombinants

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.