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.

Attenzione: consultare le schede di sicurezza (SDS) di tutte le sostanze chimiche coinvolte prima dell&#39;uso. L&#39;esperimento screening di stupefacenti prevede la sintesi e la manipolazione dei nanomateriali, che possono avere rischi aggiuntivi rispetto alla loro controparte massa. Si prega di verificare le misure di controllo necessarie per essere praticato tutto l&#39;esperimento, compreso l&#39;uso di controlli tecnici (cappa) e dispositivi di protezione individuale (DPI, ad esempio, i pantaloni di lunghezza di sicurezza, scarpe chiuse, guanti resistenti ai prodotti chimici, e occhiali di protezione). 1. Preparazione dei reagenti chimici per lo screening Drug <ol><li> Precursori per la sintesi Au NC <ol><li> Disciogliere 30 mg di soluzione di oro (III) cloruro (99,99% base oligometalli, 30 wt.% In HCl diluito) in 6,9 ml di acqua ultrapura per preparare 15 mM di oro (III) di cloruro. Attenzione: soluzione di cloruro d&#39;oro è corrosivo e irritante. Indossare corretto DPI per evitare il contatto diretto con gli occhi e la pelle. </li><li> Dissolve 74 mg di HSA o BSA in 1 ml di acqua ultrapura per preparare 74 mg / ml di proteine ​​soluzione madre. </li></ol></li><li> Soluzioni di droga <ol><li> Sciogliere la quantità desiderata di farmaco, per esempio, 1,9 mg per (a) ibuprofene, 2.8 mg di (b) warfarin, 2,3 mg di (c) fenitoina, e 1,5 mg per (d) sulfanilamide in 20 ml di DMSO per preparare 450 mM di soluzioni droga azionari. Nota: DMSO viene selezionato come solvente perché questi farmaci sono idrofobi e hanno scarsa solubilità in acqua. </li></ol></li><li> Altri reagenti <ol><li> Sciogliere 600 mg di pellet NaOH in 10 ml di acqua ultrapura per preparare 1,5 M di NaOH. Sciogliere 2.4 g di urea in 2 ml di acqua ultrapura per preparare 20 M di soluzione di urea. </li></ol></li></ol> 2. Sintesi di proteine ​​Templated Au NC <ol><li> HSA-su modelli Au NC (HSA-Au NC) <ol><li> Inserire due fiale di vetro, ognuno contenente un micro ancoretta magnetica in esso, su due separata della temperatura controllabile ancoretta magneticaRERS. </li><li> Impostare la temperatura di un agitatore magnetico a 60 ° C, ed etichetta la fiala di vetro su di essa come &quot;60 ° C&quot;; mentre l&#39;altro agitatore magnetico viene mantenuta a temperatura ambiente (RT) in cui il flacone di vetro su di esso è etichettato come &quot;RT&quot;. </li><li> Aggiungere 200 ml di soluzione di HSA, 200 ml di acqua ultrapura, e 200 ml di soluzione di oro (III) cloruro di ciascuna fiala sotto agitazione costante (impostata come 360 ​​rpm se non diversamente specificato) per consentire l&#39;incapsulamento di ioni Au all&#39;interno del modello della proteina. </li><li> 2 minuti più tardi, aggiungere 20 ml di soluzione di NaOH ad ogni flacone in modo da attivare la capacità di ridurre HSA nel formare Au NC e iniziare a registrare il tempo di reazione come 0 min. </li><li> Ogni 20 minuti, 50 ml di disegnare soluzioni da ciascuno del campione ad una piastra nera 384 pozzetti e misurare lo spettro di emissione con un lettore di micropiastre. La configurazione tipica di scansione: λ = 370 nm ex, λ em = 410-850 nm. </li><li> Arrestare il agitatore magnetico dopo 100 min. </li><li> Raffreddare i flaconi di vetro sotto l&#39;acqua corrente in un lavandino. Attenzione: le fiale di vetro sono caldi. Indossare guanti resistenti al calore per evitare di bruciare. </li><li> Tracciare gli spettri di fotoemissione a tutto il tempo per ciascun campione di acquisire la cinetica di formazione di Au NC a diverse condizioni di temperatura. </li></ol></li><li> BSA-su modelli Au NC (BSA-Au NC) <ol><li> Inserire una fiala di vetro contenente un micro ancoretta magnetica sulla sommità di un agitatore magnetico. Lasciare la temperatura come temperatura ambiente. </li><li> Miscelare 200 ml di soluzione di BSA, 200 ml di urea, e 200 ml di soluzione di oro (III) cloruro nel flaconcino di vetro sotto costante agitazione. </li><li> 2 minuti più tardi, aggiungere 20 ml di soluzione di NaOH per attivare la capacità riducente di BSA per formare Au NC e iniziare a registrare il tempo di reazione come 0 min. Misurare lo spettro di fotoemissione della miscela di reazione oraria. Nota: Lo spettro di fotoemissione di BSA-Au NC è misuratameno frequentemente perché il tasso di formazione BSA-Au NC è più lenta di quella di HSA-Au alla stessa temperatura a causa della minore efficacia di urea a svolgersi proteina. </li><li> Fermare l&#39;agitatore magnetico dopo 7 ore. Tracciare gli spettri di fotoemissione a tutti il ​​tempo di vedere la cinetica di formazione dei Au NC da urea denaturazione. </li></ol></li></ol> Screening 3. Piccolo Drug molecolare <ol><li> Schermata relativa affinità di legame di diversi farmaci piccola molecolari per HSA <ol><li> Posizionare cinque fiale di vetro, ciascuna contenente una ancoretta magnetica in esso, in cima ad una temperatura controllabile multipunto agitatore magnetico. Etichettare queste fiale come a, b, c, d, rispettivamente, per ciascun farmaco, vale a dire l&#39;ibuprofene, il warfarin, fenitoina, e sulfanilamide. Etichettare quello con HSA puro come controllo. </li><li> Aggiungere 200 ml di HSA ad ogni flacone. Aggiungere 1 ml di soluzione medicinale ai quattro fiale corrispondenti. Aggiungere 1 ml di DMSO al controllo. Accendere l&#39;agitatore e impostare la velocità di centrifuga a 360rpm e incubare per 1 ora per consentire il legame di HSA droga per completare. </li><li> 1 ora dopo, aggiungere 200 ml di acqua Milli-Q e 200 ml di soluzione di oro (III) cloruro di ciascuna fiala sotto costante agitazione. Impostare la temperatura a 60 ° C sotto costante agitazione per 10 min. Aggiungere 20 ml di NaOH 1,5 M a ciascuna fiala e iniziare a registrare il tempo di reazione come 0 min. </li><li> Disegnare rapidamente 50 ml di soluzione da ogni flaconcino a un piatto nero 384 pozzetti e misurare lo spettro di emissione (risultati etichettati come 0 min). Registra gli spettri di emissione di ciascun campione ogni 10 min. Raccogliere almeno quattro spettri a quattro tempi diversi, cioè 0, 10, 20 e 30 min. </li><li> Ripetere i punti 3.1.1 - 3.1.4 più volte per ottenere risultati con un trend costante. Arrestare il agitatore magnetico. </li><li> Tracciare la fotoemissione spettri risolta in tempo per ogni campione (a - d, e controllo). Identificare l&#39;intensità massima di tutti i campioni e complotto contro il tempo per confrontare la cinetica di formazione dei Au NC a dimodelli di proteina-droga caricata fferent. </li></ol></li><li> Misurare la costante di legame di un farmaco specifico per HSA <ol><li> Ripetere i punti 3.1.1 - 3.1.4. Sostituire la soluzione farmaco con soluzioni ibuprofene a quattro diverse concentrazioni (tra cui un campione di controllo utilizzando HSA solo senza droga carico). Etichettare il flacone di vetro secondo la concentrazione di farmaco corrispondentemente. </li><li> 10 minuti più tardi, raffreddare i flaconi di vetro sotto l&#39;acqua corrente in un lavandino. Attenzione: le fiale di vetro sono caldi. Indossare guanti resistenti al calore per evitare di bruciare. </li><li> Disegna 50 ml di soluzioni sopra di ogni fiala per un piatto nero 384 pozzetti e misurare lo spettro di emissione. </li><li> Ripetere i punti 3.2.1 - 3.2.3 altre due volte per ottenere altre due serie di risultati a diverse concentrazioni del farmaco. </li><li> Arrestare il agitatore magnetico. Tracciare gli spettri di fotoemissione e analizzare i risultati. <ol><li> Per i dati grezzi ottenuti in ciascun lotto, tracciare gli spettri di fotoemissione di ciascuncampione in software come software OriginPro e scoprire l&#39;intensità di picco. </li><li> Calcolare e tracciare l&#39;intensità di fluorescenza relativa [(I 0 - I) / I 0] contro la concentrazione del farmaco, dove io e mi riferisco a 0 l&#39;intensità di fluorescenza di ragazza NC formato in-droga caricato HSA e puro HSA, rispettivamente. </li><li> Calcolare la costante di legame inserendo i dati in un unico luogo di modello di legame utilizzando l&#39;equazione di Michaelis-Menten: Y = R max × C / (K D + C), dove R max indica il segnale di risposta massima, C è la concentrazione di ligando e K D è la costante di legame. Eseguire questo software come OriginPro. Selezionare il menu di analisi non lineare Montaggio Adattamento curva, quindi selezionare la funzione Hill Crescita / categoria sigmoidale. Nella Impostazioni: pagina di selezione funzione, fare clic su Adatta a mostrare i risultati a muro. </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.

Ci sono diversi passaggi critici che devono essere evidenziate in questo metodo. Nel protocollo di screening della affinità di legame relativo dei diversi farmaci molecolari piccoli passi 3.1.2, 3.1.3, 3.1.4 e sono fondamentali per ottenere buoni risultati mostrano tendenza coerente per la forza vincolante relativa. In queste fasi, le azioni di aggiunta di sostanze chimiche e disegno di soluzioni di reazione per la misura dovrebbe essere il più rapidamente possibile per minimizzare l&#39;effetto ritardo e la stessa se...

Albumine siero come albumina sierica umana (HSA) e albumina di siero bovino (BSA) sono la proteina più abbondante nel plasma e svolgono un ruolo fondamentale nel mantenimento della pressione osmotica del comparto sangue. Sono inoltre riconosciuti come proteine ​​di trasporto per le piccole molecole di bassa solubilità in acqua, come gli steroidi, acidi grassi, ormoni tiroidei, e una vasta gamma di farmaci. L&#39;associazione di proprietà (ad esempio, siti di legame, affinità o la forza vincolante) di queste molecole di siero albumine costituisce un tema importante nella farmacocinetica. 1-4 diversi metodi analitici sono stati sviluppati per studiare le proprietà di legame dei farmaci diversi per siero albumine, come ad esempio cristallografia a raggi X, risonanza magnetica nucleare 5,6 (NMR), 7-11 e risonanza plasmonica superficiale (SPR), 12,13 etc. Tuttavia, questi metodi sono vincolate mediante un processo di analisi noioso e che richiede tempo (ad esempio, la crescita del monocristallo per X-ray Crystallostudio grafico), requisito di attrezzature specializzate e costose (SPR), o che necessitano di marcatura isotopica costosi (NMR) per la rilevazione. E &#39;quindi altamente auspicabile sviluppare modalità alternative per lo screening piccolo farmaco molecolare in un modo veloce, straight-forward, ed economicamente efficiente. Nanocluster oro (Au NC) sono un tipo speciale di nanomateriali, che contengono diverse decine di atomi di metallo con dimensioni inferiori a 2 nm. 14-17 hanno attirato ampi interessi di ricerca a causa della loro struttura elettronica discreta e dimensione-dipendente, 18, 19 e molecolare come assorbimenti ed emissioni. 20-23 Tali proprietà materiali unici, in particolare una forte fluorescenza, hanno trovato diverse applicazioni come il rilevamento e l&#39;imaging in sistemi biologici. 24-32 Ultrasmall fluorescente Au NC può essere sintetizzato utilizzando proteine ​​funzionali, quali albumine siero, come modello. 33 In una tipica sintesi delle proteine-su modelli di<html> Au NC, una certa quantità di sali Au vengono prima incapsulati all&#39;interno della proteina e successivamente ridotta di proteina stessa. La capacità riducente della proteina è attribuita a costituente residui amminoacidici funzionali (ad esempio, tirosina) che possono essere attivati ​​aumentando il pH della soluzione ad alcalino. Dispiegarsi della struttura delle proteine ​​è considerato come un passo fondamentale per la formazione di Au NC. Questo perché in una proteina unfolded, più gruppi funzionali riducenti possono essere esposti ai sali Au incapsulati. Protein dispiegarsi può essere ottenuto mediante trattamento termico o esposizione ad agenti denaturanti. Introduzione di piccoli farmaci molecolari può anche interessare il processo di svolgimento, cioè modificando la temperatura di denaturazione punto medio e l&#39;entalpia di svolgimento. 34,35 L&#39;effetto di tutti questi fattori, a sua volta può essere riflessa dalla cinetica di formazione dei fluorescenti Au NC e si manifesta in l&#39;intensità di fluorescenza di risultanti Au NC. 36 e_content &quot;&gt; Questo video illustra il metodo di selezione della droga sintetizzando Au NC in proteine ​​albumina droga caricata ad una temperatura superiore (60 ° C) o in presenza di agenti denaturanti (ad esempio, urea). L&#39;intensità di fluorescenza della risultante Au CN è la lettura del segnale. Innanzitutto, Au NC sono sintetizzati nei modelli HSA e BSA trattati a 60 ° C o in presenza di urea per mostrare come proteina dispiegarsi (indotta mediante trattamento termico o denaturanti) influenza la cinetica di formazione di Au NC. In secondo luogo, Au NC sono sintetizzati nei modelli di proteine ​​precaricato con diversi farmaci, e l&#39;effetto della droga carico sui intensità di fluorescenza relativi dei risultante Au NC è studiato, che forniscono la misura della forza vincolante relativa. Infine, il protocollo di screening Au NC-farmaco viene modificato per misurazione quantitativa di farmaco-proteina legante costante (K D) variando il contenuto di farmaco precaricato nella proteina di una concentrazione fissa.

<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

Si dimostra un nuovo metodo di screening di farmaci per determinare l&#39;affinità di legame di piccole molecole farmaceutiche per una proteina bersaglio formando nanocluster fluorescenti oro (Au NC) all&#39;interno della proteina-droga caricata, sulla base del segnale di fluorescenza emessa dal differenziale Au NC. Proteine ​​albumina come l&#39;albumina sierica umana (HSA) e albumina sierica bovina (BSA) sono selezionati come le proteine ​​modello. Quattro piccole farmaci molecolari (ad esempio, l&#39;ibuprofene, il warfarin, fenitoina, e sulfanilamide) di diverse affinità di legame alle proteine ​​albumina sono testati. Si è constatato che il tasso di formazione di fluorescenti Au CN all&#39;interno farmaco caricata albumina in condizioni denaturanti (cioè, 60 ° C o in presenza di urea) è più lenta di quella formata nella proteina intatta (senza farmaci). Inoltre, l&#39;intensità fluorescente del CN ​​come formata è risultato essere inversamente correlato alle affinità di legame di questi farmaci alle proteine ​​albumina. In particolare,maggiore è l&#39;affinità di legame farmaco-proteina, più lenta è la velocità di formazione Au NC, e quindi una minore intensità di fluorescenza della risultante Au NC viene osservata. L&#39;intensità di fluorescenza della risultante Au NC fornisce quindi una misura semplice della forza di legame relativa di diversi farmaci testati. Questo metodo è anche estensibile per misurare la costante legame specifico farmaco-proteina (K D) semplicemente variando il contenuto droga precaricato nella proteina ad una concentrazione proteica fissa. I risultati misurati corrispondono bene con i valori ottenuti con altri metodi di prestigio ma più complicati.

Protein dispiegarsi un importante metodo per la formazione di proteina-templated Au NC perché gruppi funzionali più reattivi (per esempio, residui di tirosina di una proteina) possono essere esposti per ridurre gli ioni Au incapsulati e accelerare il tasso di formazione di Au NC così. Agenti di riscaldamento e di denaturazione esterno sono due mezzi comuni per promuovere il processo di svolgimento delle proteine. La Figura 1 mostra l&#39;effetto di riscaldamento e l&#39;aggiunta di agenti di...

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.

Una rapida e quantitativa metodo fluorimetrico per lo screening di stupefacenti piccole molecole di proteine ​​Targeting

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

Research

JoVE Journal

Bioengineering

9.9K Views.  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.

Video: 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.

Una migliore visualizzazione e analisi quantitativa degli effetti del farmaco utilizzando cellule 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 ...

Ricerca

Bioingegneria

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) per High-throughput screening farmaco antifungino

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.

Indagare Sistemi recettore-ligando del Cellulosome con sede a AFM singola molecola di Forza Spettroscopia

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.

Farmaco-indotta Sensibilizzazione della ciclasi: L&#39;ottimizzazione del dosaggio e miniaturizzazione per piccole molecole di siRNA e applicazioni di screening

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.

Rapid Mix Preparazione di bioispirati Nanoscale idrossiapatite per applicazioni biomediche

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.

Automated Analysis Contrazione di ingegneria dei tessuti umani Cuore per lo screening cardiaco Drug Safety

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 per la terapia rigenerativa iniettabile e droga di High-throughput Screening

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.

Reprimere la trascrizione genica reindirizzando macchinario cellulare con modificatori epigenetici chimici

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.

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

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;

Applicazioni per pannelli di illuminazione compatibili con microplacca 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 ...

Una rapida e quantitativa metodo fluorimetrico per lo screening di stupefacenti piccole molecole di proteine ​​Targeting

Una rapida e quantitativa metodo fluorimetrico per lo screening di stupefacenti piccole molecole di proteine Targeting