Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Piatto 20.000 differenziate cellule staminali embrionali di topo (MES) / pozzetto in piastre da 96 lisina rivestito Poly-D e così mantenere in motoneurone differenziazione terminale dei media per 5-7 giorni. 1. Compound Amministrazione e Intossicazione con BoNT / A Eseguire tutti i seguenti lavori in un recinto BSL2 per mantenere la conformità con le linee guida CDC / NIH. <ol><li> Preparare 10 mM soluzioni madre di ciascun composto libreria in 100% DMSO in polipropilene piastre da 96 pozzetti. Utilizzare lo stock 10 mM per preparare un piatto di diluizione intermedia che è 10 volte superiore alla concentrazione di retinatura desiderato. Preparare 100 uM piastra intermedia per l&#39;erogazione di 10 stock mm nella piastra a 96 pozzetti e diluire a 100 uM utilizzando terreni di coltura. Pipettare 10 ml di composti in 90 ml di media sulle cellule 11. Testare i composti: 10 uM concentrazione finale e garantire la percentuale finale di DMSO non supera 0,5%. </li><li> Eseguire tutte le vite prigionieraIES che utilizzano cellule vive e tossina attiva sotto il livello di biosicurezza 2 condizioni. Sostituire i vecchi media nelle piastre a 96 pozzetti con 80 ml di fresca supporti differenziazione terminale utilizzando una pipetta manuale a 12 canali. Effettuare l&#39;aspirazione ed erogare fasi righe per evitare l&#39;esposizione delle cellule senza supporto per l&#39;aria ambiente. </li><li> Pretrattare cellule con composti prima dell&#39;applicazione della tossina mediante aggiunta di 10 ml di 10x composti dalla piastra di origine usando una pipetta a 12 canali, seguita da miscelazione per aspirazione (due volte). Per ogni piastra a 96 pozzetti, il trattamento di due colonne di 6 pozzi con 10x DMSO diluito nei media per servire come segnale alto e comandi in bassa di segnale. La colonna senza trattamento BoNT presenta il massimo livello di tutta la lunghezza SNAP-25 (controllo segnale alto). Trattare l&#39;altra colonna con BoNT da solo (senza componenti), come il controllo del segnale basso. Utilizzare un controllo ottimo per osservare l&#39;efficacia di BoNT scissione di SNAP-25 e la sua individuazione con l&#39;anticorpo BACS (Figura 2). </li><li> Incubare le cellule con composti per 30 minuti a 37 ° C e 6% di CO 2 in incubatrice coltura cellulare. </li><li> Preparare neurotossina botulinica A da un 1 mg / fonte commerciale ml in tampone fosfato (PBS) per una finale 10x lavoro diluendo con i media differenziazione terminale. </li><li> Avviare intossicazione con l&#39;aggiunta di 10 ml di 10x BoNT / A magazzino in pozzi riconosciuto per il trattamento e basso segnale controlli utilizzando una pipetta multicanale (Figura 2). Trattare i pozzetti designati come di tenere sotto controllo con i soli mezzi. </li><li> Decontaminare tutti ware plastica e altri monouso che viene a contatto con BoNT / A durante lo studio mediante immersione in una soluzione di ipoclorito di 0,825% in acqua per almeno 20 min. Eseguire tutte le procedure di cappe di sicurezza biologica e decontaminare le aree di lavoro sia prima che dopo gli esperimenti con il 5% di detergente disinfettante più pulita, come soluzione MicroChem. </li><li> Etichettare le piastre in modo chiaro per indicare l&#39;uso di tossine e incubpiastre mangiato trattati con 1 nM / L BoNT / A per 4 ore a 37 ° C e 6% di CO 2 in un incubatore di coltura cellulare. Visualizza adeguata segnaletica per informare i colleghi che la tossina è in uso in laboratorio. </li><li> Arresto BONT / A taglio proteolitico ad opera di fissazione metanolo. Rimuovere i supporti contenenti tossine e composti da ogni pozzetto con una pipetta multicanale e scartare in soluzione di candeggina al 10%. Aggiungere il ghiaccio metanolo freddo (100 ml) direttamente a ciascun pozzetto. </li><li> Incubare le piastre per 15 minuti a temperatura ambiente (RT) per permettere il fissaggio a verificarsi. </li><li> Dopo la fissazione, di gestire in modo sicuro i reagenti di scarto (considerato neutralizzate) con livello di biosicurezza 1 protocolli ed eliminare di conseguenza. </li><li> Eliminare metanolo e utilizzare 100 ml di PBS per lavare le cellule e reidratare la loro prima della colorazione. Eseguire due ulteriori lavaggi PBS. </li><li> Dopo l&#39;ultimo lavaggio, lasciare PBS nei pozzi, piastre di tenuta con pellicola adesiva di micropiastre e conservare a 4 ° C fino al momento dell&#39;analisi. Conservare le piastre a 4 ° C for diversi giorni. </li></ol> 2. Immunostaining La procedura di immunoistochimica è un ad alta intensità di lavoro, il funzionamento multifase che comprende reagenti ripetitivi cicli di erogazione / aspirazione e la piastra vasta lavaggio che possono provocare l&#39;introduzione di una significativa variabilità intraplacca e interplate. Un approccio semi-automatico viene applicato per risparmiare il tempo del personale di laboratorio, aumentare la velocità di dosaggio, e ridurre al minimo la variabilità immunocolorazione. <ol><li> Utilizzare una stazione di lavoro automatizzata dotata di testa a 96 pozzetti in grado di trasferire volumi fino a 250 ml e integrato con un braccio robotico pinza a muoversi da laboratorio in diverse posizioni sul ponte modulare (vedi Materiali e attrezzature) per questo protocollo. <ol><li> La piattaforma modulare è progettato per ospitare diversi tipi di articoli da laboratorio e può supportare fino a 11 elementi in qualsiasi momento. Il gestore di micropiastre è dotato di un doppio scomparto micropiastre impilatore per trattenere emanipolare fino a 50 piastre per ciclo. </li><li> La stazione di lavoro automatizzata si trova all&#39;interno di un armadio biosicurezza di classe II progettato per apparecchiature per l&#39;automazione di laboratorio per fornire la sterilità e sicurezza. Per immunostaining automatizzato, il ponte è disposta come mostrato in Figura 3 per consentire l&#39;accesso dei reagenti secondo necessità e senza intervento umano. </li></ol></li><li> Piastre di pre-carico contenenti cellule fissate nel caricatore piastra e trasferimento al ponte come necessario per le operazioni di manipolazione dei liquidi. Usare la testa 96 pipetta dotato di 250 punte microlitri per aspirare PBS dai pozzi fino a 10 ml. Permeabilize membrane cellulari per facilitare il legame dell&#39;anticorpo a bersagli intracellulari con tampone permeabilizzazione (0.1% Triton X-100). Distribuire buffer di permeabilizzazione (100 microlitri) in ciascun pozzetto prima di tornare le piastre secondo magazzino di stoccaggio del raccoglitore micropiastra per 15 minuti di incubazione a temperatura ambiente (RT). </li><li> Rimuovere buffer di permeabilizzazione e pelavaggio rform piastra con PBS (due volte) come descritto in precedenza al punto 1.13. </li><li> Dopo l&#39;ultimo ciclo di lavaggio, rimuovere il tampone PBS e dispensare 100 ml di tampone bloccante contenente siero di cavallo 1% e lo 0,1% di Tween 20 in PBS in tutti micropiastre prima di restituirli alla rivista piastra per 1 ora di incubazione a RT. </li><li> Utilizzare due anticorpi primari per immunoistochimica: un anti-topo anticorpi BACS policlonale di coniglio, per il rilevamento di piena lunghezza anticorpi anti-III tubulina SNAP-25 e un anticorpo monoclonale del mouse per il rilevamento dei neuroni (vedi Materiali e attrezzature). Dopo 60 min di incubazione, rimuovere tampone bloccante ed erogare 50 ml di 4ug / ml di anticorpo BACS e 0,5 mg / ml di III-tubulina in tampone di bloccaggio in tutti i piatti. </li><li> Incubare per 1 ora a RT rimuovere gli anticorpi primari e lavata 3 volte con 100 ml di PBS contenente 0,05% Tween (PBST). </li><li> Utilizzare un IgG anti-topo marcato con Alexa Fluor 647 per visualizzare le IgG-III tubulina e anti-coniglio taggati con Alexa Fluor 568 Per visualizzare l&#39;anticorpo BACS. Preparare entrambe anticorpi come una diluizione 2 ug / ml in tampone bloccante ed erogare 50 ml di miscela in ogni pozzetto. NOTA: Gli anticorpi secondari selezionati per immunoistochimica sono etichettati con differenti fluorofori per consentire il rilevamento della loro luce fluorescente emessa a lunghezze d&#39;onda diverse che utilizzano canali diversi all&#39;interno del rivelatore dell&#39;Alto Imager contenuti. </li><li> Incubare per 1 ora a RT. Aspirare gli anticorpi secondari e lavare 3 volte con 100 ml di PBST. </li><li> Dopo l&#39;ultimo lavaggio, aggiungere 100 ml di PBS contenente colorante Hoechst (32 micron) per colorare il DNA per il rilevamento di nuclei. </li><li> Sigillare le piastre con una pellicola impermeabile leggero per proteggere i fluorofori da photobleaching. Le piastre possono essere conservati a 4 &#160; ° C per settimane senza una significativa perdita di segnale. </li></ol> 3. Imaging NOTA: Eseguire l&#39;acquisizione di immagini con alto contenuto di Imaging SysTEM (vedi Materiali e attrezzature). <ol><li> Specifiche di contenuti ad alta definizioni Imager: <ol><li> Selezionare il tipo di piatto, struttura, numero di campi, obiettivo: Greiner u chiaro, formato 96 bene, 16, rispettivamente, 20X acqua. </li><li> Selezionare i canali: nucleo di eccitazione EX: 405 nm come canale 4; citoplasma EX: 644 nm come canale 2; antigene specifico EX canale anticorpi: 561 nm come canale 3 e Impostazione potenza di eccitazione di ogni laser, esperimento Imposta come 2 esposizioni, Esposizione 1 con una eccitazione a 644 nm, Esposizione 2 con due eccitazioni a 405 nm e 561 nm. Selezionare binning come BIN2. </li><li> Utilizzare pozzetti di controllo elevate per l&#39;ottimizzazione dell&#39;esposizione con la massima intensità come canale di SNAP-25 e pozzetti di controllo bassi per intensità minima. </li><li> Regolare l&#39;esposizione, in modo che l&#39;intensità di ciascun canale è circa la metà del livello di saturazione (2.000-2.500 unità relative). </li><li> Identificare il piano ottimale Z sul canale della maschera cellulare utilizzando delta script di correzione. See Figura 5 per risultati dopo immunoistochimica e di imaging. Esportare i dati al server in cui il software di analisi delle immagini risiede. </li></ol></li></ol> 4. Analisi dell&#39;immagine (Figura 6) NOTA: La seguente procedura descrive l&#39;applicazione degli algoritmi software Columbus. <ol><li> Selezionare un algoritmo appropriato per gli oggetti primari (nuclei) segmento. Se necessario, regolare la soglia di fondo e parametri di contrasto. Il software Columbus offre 4 metodi di rilevamento nuclei. Selezionare il metodo che i nuclei segmento con precisione per un controllo a vista nuclei segmentati (in F metodo IGURA 6a M è selezionato). </li><li> Selezionare l&#39;algoritmo di neuriti (CSIRO neuriti Analisi 2) per segmentare le neuriti Se necessario, regolare i parametri di soglia di sfondo e il contrasto per rimuovere sfondo. Vedere la Figura 6b per i parametri utilizzati per la rilevazione neuriti. </li><li> Identificare le regioni neuriti (segmenti neuriti) based oggetti secondari (neuriti) utilizzando un&#39;espansione pixel -3 del canale β-tubulina III (Alexa farina 640) come maschera (figura 6c). </li><li> Calcolare l&#39;intensità dei canali di segnale (SNAP-25, (Alexa farina 568) per la regione neuriti e il canale tubulina β-III (Figura 6d e 6e). </li><li> Selezionare i parametri desiderati per l&#39;esportazione, come la lunghezza dei neuriti, intensità medie di SNAP-25, β-tubulina III, numero di nuclei, numero di segmento neuriti, ecc (Figura 6f). </li><li> Piastre di trasferimento da impilatori piastra utilizzando il robot e carico in alto Imager contenuti per l&#39;acquisizione delle immagini. </li></ol> 5. Analisi dei dati: Per valutare la robustezza del test progettato, calcolare i seguenti parametri dell&#39;esperimento base piastriforme. <ol><li> Del segnale (S) a priorità bassa (B): S / B = intensità del segnale di controllo ad alta media) / media intensità del controllo di segnale basso </li> &lt;li&gt; Coefficiente di variazione (CV) del segnale e lo sfondo (per misurare l&#39;uniformità del segnale specifico): CV = (deviazione standard (SD) / medio di campione) * 100 (%), per determinare la variabilità nella gestione dei liquidi, reagenti, ecc </li><li> Signal to Noise: S = (media intensità del segnale - intensità media del controllo segnale basso) / SD del controllo ottimo <ol><li> Calcolare il fattore Z &#39;intossicazione di una metà di una piastra a 96 pozzetti ed una intossicazione finto dell&#39;altra metà. Z &#39;= 1 - 3 * (SD del controllo segnale alto + SD del controllo segnale basso) / (media del controllo segnale alto - medio del controllo segnale basso). Per l&#39;ulteriore analisi dei dati di screening, normalizzare alcuni parametri prime primarie su una piastra di base per permettere il confronto tra le piastre e tra i diversi giorni di esperimenti. </li></ol></li><li> Percentuale di scissione, riflettendo la riduzione del segnale associato a lunghezza completa SNAP-25 rilevata dal anticorpo specifico (BACS): Cleavage% = 100% * (mean intensità del controllo elevato rapporto segnale - intensità di campione) intensità del controllo segnale alto / medio. </li><li> Percentuale di inibizione della scissione:% di inibizione = 100% * (intensità di campione - media intensità del controllo di segnale basso) / (media intensità del segnale di controllo ad alta - intensità del controllo di segnale basso media). </li></ol>

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacophore+Refinement+Guides+the+Rational+Design+of+Nanomolar-Range+Inhibitors+of+the+Botulinum+Neurotoxin+Serotype+A+Metalloprotease.">Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.</a> ACS medicinal chemistry letters. 1, 301-305 (2010).</li><li>Kiris, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem cell research. 6, 195-205 (2011).</li><li>Pellett, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+targeting,+internalization,+and+biological+activity+of+a+recombinant+atoxic+derivative+of+botulinum+neurotoxin+A.">Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.</a> Biochemical and biophysical research communications. 405, 673-677 (2011).</li><li>McNutt, P., Celver, J., Hamilton, T., Mesngon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+neurons+are+a+novel,+highly+sensitive+tissue+culture+platform+for+botulinum+research.">Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.</a> Biochemical and biophysical research communications. 405, 85-90 (2011).</li><li>Fischer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimodal+modulation+of+the+botulinum+neurotoxin+protein-conducting+channel.">Bimodal modulation of the botulinum neurotoxin protein-conducting channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 1330-1335 (2009).</li></ol>

Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

L&#39;elevata potenza di neurotossine botuliniche e la relativa facilità del loro militarizzazione ha portato nella loro classificazione come categoria A (priorità più alta) agenti biothreat dai Centri statunitensi per il controllo e la prevenzione delle malattie. Purtroppo, non ci sono FDA ha approvato terapie per contrastare BoNT intossicazione dopo la tossina è stato interiorizzato dai motoneuroni. Qualsiasi meccanismo di druggable che promuove il recupero neuronale da BoNT intossicazione potrebbe portare allo sv...

Il batterio Clostridium botulinum produce neurotossina botulinica, una delle più potenti tossine biologiche conosciute all&#39;uomo 1. Ci sono 7 diversi sierotipi BoNT (BoNT / AG). BoNT / A-Gall indurre paralisi a livello della giunzione neuromuscolare a causa di complessi SNARE proteolisi 2,3. SNARE proteolisi impedisce neurotrasmettitore vescicole-fusione delle membrane e quindi blocchi neurotrasmettitore 4. Il target SNARE specifica dipende dal particolare sierotipo BoNT coinvolti nel processo intossicazione. BoNT / A e BoNT / E fendere SNAP-25, mentre BoNT / C scinde sia SNAP-25 e sintaxina 5. I restanti sierotipi sinaptobrevina fendere (chiamato anche vescicole associata proteina di membrana (VAMP). BoNT / A è stato scelto per lo sviluppo del test in quanto è responsabile di una percentuale elevata di naturale il botulismo e ha la più lunga durata d&#39;azione 6. Sviluppo di piccole molecole terapie contro BoNT / A è un obiettivo importante peril nostro programma di scoperta di nuovi farmaci e ha utilizzato metodi tradizionali di obiettivi per identificare inibitori sito proteolitici attivo 7, 8-10. Tuttavia, la creazione di inibitori del sito attivi con attività ad ampio spettro contro i sierotipi multipli e sull&#39;efficacia dopo l&#39;esposizione sarà probabilmente difficile. Abbiamo quindi implementato un approccio innovativo, fenotipica droga scoperta che utilizza BoNT SNAP-25 scissione come un endpoint funzionale per identificare piccole molecole in grado di bloccare BoNT-mediata intossicazione del motoneurone. SNAP-25 è necessario per il rilascio dei neurotrasmettitori, come la degradazione di SNAP-25 è predittivo di paralisi e letalità in vivo. Ad esempio, lo screening a base di cellule potrebbe portare alla scoperta di nuovi modulatori di fattori cellulari responsabili della tossina inattivazione o l&#39;inibizione delle vie di tossine all&#39;interno delle cellule bersaglio. Un fattore importante nello sviluppo test fenotipico è la selezione di fisiologicamente rilevanti modelli biologici. We ed altri hanno descritto il mouse staminali embrionali (ES) derivati ​​dalle cellule neuroni motori che ricapitolano il carattere immunologico dei motoneuroni primari, tra cui l&#39;espressione della SNAP-25 11- 13. È importante sottolineare che questi sistemi cellulari sono molto sensibili alle BoNT / A intossicazione e dimostrano scissione dose-dipendente della SNAP-25 in risposta a concentrazioni crescenti di tossina 11,12. I neuroni motori differenziati sono prodotti anche in quantità sufficiente per l&#39;analisi basata piastra throughput elevato e hanno permesso la progettazione di una serie di saggi cellulari. Il test fenotipico è un metodo di immunofluorescenza che utilizza due anticorpi distinti per quantificare scissione di endogeno espresso pieno lunghezza SNAP-25 durante la BoNT / A intossicazione della cultura del mouse motoneurone. Un terminale carbossilico BoNT / A (BACS) anticorpi scissione-sensibile che riconosce solo a figura intera SNAP-25 permette di valutare BoNT / A proteolisi mediata di SNAP-25espressione in neuroni motori del mouse 10. Un diagramma schematico del test HCI è rappresentato in figura 1.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
</table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

I dati di controllo alta e bassa create due distinte popolazioni con la differenza delle due mediane superiori a 3 deviazioni standard (Figura 7A). L&#39;obiettivo del processo di screening è quello di trovare i composti all&#39;interno della popolazione campione con valori più vicini alla popolazione di controllo positivo, ipotizzando una distribuzione normale nella popolazione campione (Figura 7B, (i)). Punti di dati che sono 3 deviazioni standard oltre la media sono considerati sta...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Un alto dosaggio Imaging contenuti per l&#39;identificazione di neurotossina botulinica inibitori

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K Views.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Video: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un saggio Slice organotipica per High-Resolution Time-Lapse Imaging della migrazione neuronale nel cervello postnatale

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Ricerca

Neuroscienze

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Ad alta risoluzione magnetica funzionale per immagini Metodi risonanza per mesencefalo umano

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imaging funzionale di Auditory Cortex in gatti adulti utilizzando ad alto campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Isolamento e quantificazione della neurotossina botulinica da matrici complesse usando il BoTest Matrix saggi

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta risoluzione<em&gt; In Vivo</em&gt; Manuale Protocollo Segmentazione for Human ippocampale Sottocampi Utilizzando 3T Risonanza Magnetica

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Non invasiva parenchimale, vascolare e metabolica ad alta frequenza ad ultrasuoni e fotoacustico Rat profonda Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Ad alta risoluzione strutturale Risonanza Magnetica del subcortex Umana<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sviluppo di analisi per la quantificazione alto contenuto di formazione aggregata di Sod1 mutante della proteina in cellule viventi

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.