Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Attention: S&#39;il vous plaît consulter les fiches de données de sécurité (FDS) de tous les produits chimiques impliqués avant utilisation. L&#39;expérience de criblage de médicaments implique la synthèse et la manipulation des nanomatériaux, qui peut avoir des risques supplémentaires par rapport à leur homologue vrac. S&#39;il vous plaît assurer que toutes les mesures de contrôle nécessaires pour être pratiquées pendant toute l&#39;expérience, y compris l&#39;utilisation des contrôles d&#39;ingénierie (hottes) et équipement de protection individuelle (EPI, par exemple, un pantalon long de la sécurité, des chaussures fermées, des gants résistant aux produits chimiques, et des lunettes de sécurité). 1. Préparation des réactifs chimiques pour le dépistage de drogues <ol><li> Précurseurs pour la synthèse Au CN <ol><li> Dissoudre 30 mg de solution d&#39;or (III) de chlorure (99,99% métaux traces de base, 30 en poids.% Dans HCl dilué) dans 6,9 ml d&#39;eau ultra pure pour préparer 15 mM d&#39;or (III) de la solution de chlorure. Attention: solution de chlorure d&#39;or est corrosif et irritant. Porter l&#39;EPI approprié pour éviter le contact direct avec les yeux et la peau. </li><li> Dissolve 74 mg de HSA ou BSA dans 1 ml d&#39;eau ultra pure pour préparer 74 mg / ml de solution de protéines de stock. </li></ol></li><li> Solutions médicamenteuses <ol><li> Dissoudre la quantité désirée de médicament, par exemple, 1,9 mg de (a) l&#39;ibuprofène, 2,8 mg de (b) la warfarine, 2,3 mg de (c) la phénytoïne, et 1,5 mg de (d) sulfanilamide dans 20 pi de DMSO pour préparer 450 mM de solutions mères de médicament. Remarque: Le DMSO est choisi comme solvant parce que ces médicaments sont hydrophobes et ont une faible solubilité dans l&#39;eau. </li></ol></li><li> D&#39;autres réactifs <ol><li> Dissoudre 600 mg de NaOH en pastilles dans 10 ml d&#39;eau ultra-pure pour préparer 1,5 M de solution de NaOH. Dissoudre 2,4 g d&#39;urée dans 2 ml d&#39;eau ultra pure pour préparer 20 M de solution d&#39;urée. </li></ol></li></ol> 2. synthèse de la protéine-Templated Au CN <ol><li> HSA-templated Au CN (HSA-Au CN) <ol><li> Placez deux flacons en verre, chacune contenant une barre d&#39;agitation magnétique micro en elle, à deux température séparé agitation magnétique contrôlableRER. </li><li> Régler la température d&#39;un agitateur magnétique à 60 ° C, et l&#39;étiquette du flacon de verre sur le dessus de celui-ci en tant que &quot;60 ° C&quot;; tandis que l&#39;autre agitateur magnétique est maintenu à température ambiante (TA) lorsque le flacon de verre sur le dessus de celui-ci est marqué comme &quot;RT&quot;. </li><li> Ajouter 200 ul de solution de HSA, 200 ul d&#39;eau ultrapure, et 200 ul d&#39;une solution d&#39;or (III) de chlorure de chaque flacon sous agitation constante (défini comme 360 ​​tours par minute, sauf indication contraire) pour permettre l&#39;encapsulation de Au ions à l&#39;intérieur du gabarit de la protéine. </li><li> 2 minutes plus tard, ajouter 20 ul de solution de NaOH à chaque flacon de manière à activer la capacité réductrice de la HSA dans la formation Au CN et commencer à enregistrer la durée de réaction à 0 min. </li><li> Chaque 20 min, tirer 50 pl de solutions à partir de chacun de l&#39;échantillon d&#39;une plaque noire de 384 puits et mesurer le spectre d&#39;émission avec un lecteur de microplaque. La configuration typique de balayage: λ ex = 370 nm, λ em = 410-850 nm. </li><li> Arrêtez l&#39;agitateur magnétique après 100 min. </li><li> Refroidir les flacons de verre sous l&#39;eau courante dans un évier. Attention: Les flacons de verre sont chauds. Porter des gants résistants à la chaleur pour éviter de brûler. </li><li> Tracer les spectres de photoémission en tout temps pour chaque échantillon d&#39;acquérir la cinétique de formation de Au CN à différentes conditions de température. </li></ol></li><li> BSA-templated Au CN (BSA-Au CN) <ol><li> Placez un flacon en verre contenant une barre d&#39;agitation magnétique micro sur le dessus d&#39;un agitateur magnétique. Laisser la température que la température ambiante. </li><li> Mélanger 200 ul de solution de BSA, 200 ul d&#39;urée, et 200 pl de solution d&#39;or (III) de chlorure dans le flacon en verre sous agitation constante. </li><li> 2 minutes plus tard, ajouter 20 ul de solution de NaOH à activer la capacité de réduction de BSA pour former Au CN et commencer à enregistrer la durée de réaction à 0 min. Mesurer le spectre du mélange de réaction de photoémission horaire. Remarque: Le spectre de BSA-Au CN photoémission est mesuréemoins fréquemment parce que le taux de BSA-Au CN formation est plus lente que celle de la HSA-Au à la même température en raison de moins d&#39;efficacité de l&#39;urée pour déplier la protéine. </li><li> Arrêtez l&#39;agitateur magnétique après 7 h. Tracer les spectres de photoémission en tout temps pour voir la cinétique de formation de Au CN par l&#39;urée dénaturation. </li></ol></li></ol> Projection 3. Petit Drug moléculaire <ol><li> Écran affinité de liaison relative des différents médicaments à petites moléculaires à HSA <ol><li> Placez cinq flacons de verre, chacun contenant une barre d&#39;agitation magnétique en elle, au-dessus d&#39;une température contrôlable multipoint agitateur magnétique. Marquez ces flacons comme A, B, C et D, respectivement, pour chaque médicament, à savoir l&#39;ibuprofène, la warfarine, la phénytoïne et sulfanilamide. Étiqueter celui avec HSA pur comme contrôle. </li><li> Ajouter 200 pi de HSA à chaque flacon. Ajouter 1 pi de solution de médicament à quatre flacons correspondants. Ajouter 1 pi de DMSO à la commande. Allumez l&#39;agitateur et régler la vitesse de rotation à 360rpm et incuber pendant 1 heure pour permettre la liaison à la HSA médicament pour terminer. </li><li> 1 h plus tard, ajouter 200 ul d&#39;eau Milli-Q et les 200 ul de solution d&#39;or (III) de chlorure de chaque flacon sous agitation constante. Régler la température à 60 ° C sous agitation constante pendant 10 min. Ajouter 20 ul de NaOH 1,5 M dans chaque flacon et commencer à enregistrer la durée de réaction à 0 min. </li><li> Dessiner rapidement 50 pi de solution de chaque flacon dans une plaque noire de 384 puits et mesurer le spectre d&#39;émission (résultats étiquetés comme 0 min). Enregistrer les spectres d&#39;émission de chaque échantillon toutes les 10 min. Recueillir au moins quatre spectres à quatre fois différente, qui est de 0, 10, 20, et 30 min. </li><li> Répétez les étapes 3.1.1 - 3.1.4 à plusieurs reprises pour obtenir des résultats avec une tendance constante. Arrêtez l&#39;agitateur magnétique. </li><li> Tracer les spectres de photoémission résolue en temps pour chaque échantillon (A - D, et de contrôle). Identifier l&#39;intensité du pic de tous les échantillons et complot contre le temps de comparer la cinétique de formation de Au CN en différents modèles de protéines chargés en médicament. </li></ol></li><li> Mesure de la constante de liaison d&#39;un médicament spécifique pour HSA <ol><li> Répétez les étapes 3.1.1 - 3.1.4. Remplacer la solution de médicament avec des solutions d&#39;ibuprofène à quatre concentrations différentes (y compris un échantillon de contrôle utilisant HSA seulement sans charge de médicament). Marquer le flacon en verre en fonction de la concentration du médicament de manière correspondante. </li><li> 10 min plus tard, refroidir les flacons de verre sous l&#39;eau courante dans un évier. Attention: Les flacons de verre sont chauds. Porter des gants résistants à la chaleur pour éviter de brûler. </li><li> Dessinez 50 pi des solutions ci-dessus de chaque flacon dans une plaque noire de 384 puits et mesurer le spectre d&#39;émission. </li><li> Répétez les étapes 3.2.1 - 3.2.3 deux fois de plus d&#39;obtenir deux autres ensembles de résultats à différentes concentrations de médicaments. </li><li> Arrêtez l&#39;agitateur magnétique. Tracer les spectres de photoémission et analyser les résultats. <ol><li> Pour les données brutes obtenues dans chaque lot individuel, tracer le spectre de photoémission de chaqueéchantillon dans les logiciels tels que les logiciels OriginPro et découvrir l&#39;intensité maximale. </li><li> Calculer et tracer l&#39;intensité de fluorescence relative [(I 0 - I) / I 0] contre la concentration du médicament, où je et je 0 réfère à l&#39;intensité de fluorescence de Au CN formée dans la drogue chargé HSA et pure HSA, respectivement. </li><li> Calcul de la constante de liaison en ajustant les données à un modèle de liaison à site unique en utilisant l&#39;équation de Michaelis-Menten: Y = R max × C / (K D + C), où R max indique le signal de réponse maximale, C est la concentration du ligand D et K est la constante de liaison. Effectuez cette dans des logiciels tels que OriginPro. Sélectionnez le menu Analyse non linéaire d&#39;adaptation Curve Fit, puis sélectionnez la fonction Hill, de croissance / catégorie Sigmoidal. Sur les paramètres: la page de sélection de fonction, cliquez sur Ajuster pour montrer les résultats ajustés. </li></ol></li></ol></li></ol>

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S., Montich, G. G., Fidelio, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

The authors have nothing to disclose.

Il ya plusieurs étapes critiques qui doivent être mis en évidence dans cette méthode. Dans le protocole de criblage de l&#39;affinité de liaison relative des différents médicaments à petites moléculaires, les étapes 3.1.2, 3.1.3, 3.1.4 et sont essentiels pour obtenir de bons résultats montrant tendance constante pour la force relative de liaison. Dans ces étapes, les actions de l&#39;ajout de produits chimiques et de l&#39;élaboration de solutions de réaction pour la mesure doivent être aussi rapidement q...

Les albumines sériques telles que l&#39;albumine de sérum humain (HSA) et l&#39;albumine de sérum bovin (BSA) sont la protéine la plus abondante dans le plasma et jouent un rôle vital dans le maintien de la pression osmotique du compartiment sang. Ils sont également reconnus comme protéines porteuses pour les petites molécules de faible solubilité dans l&#39;eau, tels que les stéroïdes, les acides gras, les hormones thyroïdiennes, et une grande variété de médicaments. La propriété de liaison (par exemple, les sites de liaison, d&#39;affinité ou force de liaison) de ces molécules au sérum albumines forme un sujet important de la pharmacocinétique. 1-4 Plusieurs méthodes analytiques ont été développées pour étudier les propriétés de liaison de différents médicaments à la sérum albumine, comme cristallographie aux rayons X, 5,6 résonance magnétique nucléaire (RMN), 7-11 et la résonance plasmonique de surface (SPR), 12,13, etc. Cependant, ces méthodes sont limitées soit par un processus d&#39;analyse fastidieux et prend du temps (par exemple, la croissance de monocristal pour cristallographique aux rayons Xétude graphique), l&#39;exigence de l&#39;équipement spécialisé et coûteux (SPR), ou dans le besoin de marquage isotopique coûteuse (RMN) pour la détection. Il est donc hautement souhaitable de développer des moyens alternatifs pour les petites criblage moléculaire de la drogue d&#39;une manière rapide, straight-forward, et rentable. Nanoclusters de l&#39;or (Au ​​CN) sont un type spécial de nanomatériau qui contiennent plusieurs dizaines d&#39;atomes de métal avec des tailles inférieures à 2 nm. 14-17 Ils ont attiré de vastes intérêts de recherche en raison de leur structure électronique discrète et dépendant de la taille, 18, 19 et moléculaire, comme des absorptions et des émissions. 20-23 Ces propriétés des matériaux uniques, en particulier la fluorescence forte, ont trouvé diverses applications telles que la détection et l&#39;imagerie dans des systèmes biologiques. 24-32 Ultrasmall fluorescent Au CN peut être synthétisé en utilisant des protéines fonctionnelles, telles que les albumines sériques, comme matrice. 33 Dans une synthèse typique de protéine sur matrice de<html> Au CN, une certaine quantité de sels sont des Au premier encapsulé à l&#39;intérieur de la protéine et par la suite réduite de la protéine elle-même. Le pouvoir réducteur de la protéine constituant est attribuée à des résidus d&#39;acides aminés fonctionnels (par exemple, tyrosine) qui peuvent être activés en augmentant le pH de la solution à alcalin. Déroulement de la structure de protéine est considérée comme une étape critique pour la formation de Au CN. En effet, dans une protéine dépliée, plusieurs groupes fonctionnels réducteurs peuvent être exposés à l&#39;UA sels encapsulés. Dépliage de la protéine peut être obtenue par traitement thermique ou exposition à des agents dénaturants. Introduction de médicaments à petites moléculaires peut également affecter le processus de déroulement, à savoir la modification de la température de dénaturation milieu et l&#39;enthalpie de déroulement. 34,35 L&#39;effet de tous ces facteurs, à son tour, peut être réfléchie par la cinétique de formation de fluorescente Au CN et manifesté dans l&#39;intensité de fluorescence des résultantes Au CN. 36 e_content &quot;&gt; Cette vidéo montre le procédé de criblage de médicaments par synthèse Au CN en protéines d&#39;albumine médicament chargé à une température élevée (60 ° C) ou en présence d&#39;agents dénaturants (par exemple urée). L&#39;intensité de fluorescence de la résultante Au CN est la lecture du signal. Tout d&#39;abord, Au-CN sont synthétisés dans HSA et BSA modèles traités à 60 ° C ou en présence d&#39;urée pour montrer comment la protéine dépliage (induite par un traitement thermique ou dénaturants) affecte la cinétique de formation de Au CN. Deuxièmement, Au CN sont synthétisés dans les modèles de protéines préchargés avec différents médicaments, et l&#39;effet de chargement de médicament sur les intensités relatives de fluorescence de résultante Au CN est étudié, qui fournissent la mesure de la force relative de liaison. Enfin, le protocole de dépistage Au NC-médicament est modifiée pour la mesure quantitative de protéine-médicament constante de liaison (K D) en faisant varier la teneur en médicament préchargée dans la protéine d&#39;une concentration fixe.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

Nous démontrons une nouvelle méthode de criblage de médicaments pour la détermination de l&#39;affinité de liaison de petites molécules de médicament à une protéine cible par formation d&#39;nanoclusters fluorescentes d&#39;or (Au CN) au sein de la protéine de médicament chargé, sur la base du signal de fluorescence différentielle émis par le Au CN. Des protéines telles que l&#39;albumine de sérum albumine humaine (HSA) et l&#39;albumine de sérum bovin (BSA) sont sélectionnées comme les protéines du modèle. Quatre petits médicaments moléculaires (par exemple, l&#39;ibuprofène, la warfarine, la phénytoïne et sulfanilamides) de différentes affinités de liaison aux protéines d&#39;albumine sont testés. Il a été constaté que le taux de formation de fluorescence Au CN à l&#39;intérieur de la protéine d&#39;albumine de médicament chargée dans des conditions de dénaturation (par exemple, 60 ° C ou en présence d&#39;urée) est plus lente que celle formée dans la protéine intacte (sans médicament). En outre, l&#39;intensité de fluorescence du CN comme formé se trouve être inversement corrélée aux affinités de liaison de ces médicaments contre les protéines d&#39;albumine. En particulier,plus l&#39;affinité de liaison protéine-médicament, le ralentissement de la vitesse de formation Au CN, et donc une intensité de fluorescence inférieure de la résultante Au CN est observée. L&#39;intensité de fluorescence de la résultante Au CN fournit donc une mesure simple de la force de liaison relative des différents médicaments testés. Cette méthode est également extensible pour mesurer la constante de liaison spécifique médicament-protéine (K D) en faisant simplement varier la teneur en médicament préchargée dans la protéine à une concentration en protéine déterminée. Les résultats mesurés correspondent bien avec les valeurs obtenues en utilisant d&#39;autres méthodes de prestige, mais plus compliquées.

Dépliement des protéines est une procédure importante pour la formation de protéines templated Au CN parce que les groupes fonctionnels plus réactifs (par exemple, des résidus de tyrosine) d&#39;une protéine peuvent être exposés à réduire les ions Au encapsulés et donc accélérer le rythme de Au CN de formation. De chauffage et de dénaturation externe agents sont deux moyens communs pour promouvoir le processus de déroulement des protéines. La figure 1 montre l&#39;effet de la c...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Un rapide et quantitative Méthode fluorimétrique pour Small Molecule dépistage du médicament protéique Ciblage

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

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JoVE Journal

Bioengineering

9.9K vues.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Vidéo: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Recherche

Bio-ingénierie

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans biofilm Chip (Ca BChip) pour criblage à haut débit des médicaments antifongiques

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Enquêter sur les systèmes récepteur-ligand de la cellulosome avec base-AFM unique molécule Spectroscopie de Force

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensibilisation à la drogue induit des adénylylcyclase: Rationalisation de dosage et la miniaturisation des petites molécules et siARN préalable Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Mix rapide Préparation de bioinspirée Nanoscale hydroxyapatite pour applications biomédicales

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automatisé Contraction L&#39;analyse des tissus humains Engineered cardiaque pour le dépistage de la sécurité cardiaque médicaments

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D pour thérapie régénérative Injectable et de dépistage des drogues de haut débit

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Réprimer la Transcription du gène en redirigeant la machinerie cellulaire avec des modificateurs chimiques épigénétiques

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purification et analyse d'un anticorps monoclonal à partir de cellules ovaires de hamster chinois à l'aide d'un système automatisé de microbioréacteur

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Applications pour les panneaux d'éclairage Microplate-Compatible open Source

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...