Name: a high content imaging assay for identification of botulinum neurotoxin inhibitors
Uploaded: 2014-11-14T13:00:00
Description: Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

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 p...

<ol>
	<li>Lamanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+most+poisonous+poison.">The most poisonous poison.</a> Science. 130, 763-772 (1959).</li><li>Dover, N., Barash, J. R., Hill, K. K., Xie, G., Arnon, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Characterization+of+a+Novel+Botulinum+Neurotoxin+Type+H+Gene.">Molecular Characterization of a Novel Botulinum Neurotoxin Type H Gene.</a> J. Infect. Dis. , (2013).</li><li>Hakami, R. M., Ruthel, G., Stahl, A. M., Bavari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaining+ground:+assays+for+therapeutics+against+botulinum+neurotoxin.">Gaining ground: assays for therapeutics against botulinum neurotoxin.</a> Trends in microbiology. 18, 164-172 (2010).</li><li>Yersin, 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=Interactions+between+synaptic+vesicle+fusion+proteins+explored+by+atomic+force+microscopy.">Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8736-8741 (2003).</li><li>Dong, M., Tepp, W. H., Johnson, E. A., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+sensors+to+detect+botulinum+neurotoxin+activity+in+vitro+and+in+living+cells.">Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 14701-14706 (2004).</li><li>Stahl, A. M., Adler, M., and, C. B. M., Gilfillan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+botulism+therapeutic+product+development+in+the+Department+of+Defense.">Accelerating botulism therapeutic product development in the Department of Defense.</a> Drug Development Research. 70, 303-326 (2009).</li><li>Nuss, J. 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=Development+of+cell-based+assays+to+measure+botulinum+neurotoxin+serotype+A+activity+using+cleavage-sensitive+antibodies.">Development of cell-based assays to measure botulinum neurotoxin serotype A activity using cleavage-sensitive antibodies.</a> Journal of biomolecular screening. 15, 42-51 (2010).</li><li>Opsenica, I., 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=A+chemotype+that+inhibits+three+unrelated+pathogenic+targets:+the+botulinum+neurotoxin+serotype+A+light+chain,+P.+falciparum+malaria,+and+the+Ebola+filovirus.">A chemotype that inhibits three unrelated pathogenic targets: the botulinum neurotoxin serotype A light chain, P. falciparum malaria, and the Ebola filovirus.</a> Journal of medicinal chemistry. 54, 1157-1169 (2011).</li><li>Opsenica, I., 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=The+synthesis+of+2,5-bis(4-amidinophenyl)thiophene+derivatives+providing+submicromolar-range+inhibition+of+the+botulinum+neurotoxin+serotype+A+metalloprotease.">The synthesis of 2,5-bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease.</a> European journal of medicinal chemistry. 53, 374-379 (2012).</li><li>Nuss, J. 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>

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.

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			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
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			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
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			<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>
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			<td height="21" style="height:21px;width:255px;">PlateStack</td>
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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 ...

Research

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Neuroscience

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

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 ...

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High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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

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