Wir erkennen an, finanzielle Unterstützung von CPRIT (RP100119) und der Welch Foundation (I-1665).

Entire Protokoll dauert ca. 4 Tage. 1. Vorbereitung der Zellen für siRNA Transfektion und Reporter Wir werden hier vorstellen ein Protokoll für die Abfrage von siRNA-Bibliotheken mit einem kleinen Satz von siRNAs (384 verschiedene siRNA-Pools Array in 96-Well-Format mit jedem Pool, bestehend aus 4x siRNAs ein einzelnes Gen) aus einer genomweiten siRNA-Bibliothek zu illustrativen Zwecken. Für diese besondere Studie werden wir nutzen HCT116 Zellen, die abweichende Wnt-Signalisierung und Kras ausstellen, und p53-Aktivität. Die geeignete Zelllinie in Studien bei Abfragen andere zelluläre Prozesse von Interesse gerichtet sind, müssen identifiziert und bewertet werden in ähnlicher Weise. <ol><li> Waschen 5 x 10 cm 2 Platten aus 80-90% konfluenten Zellen mit PBS HCT116 dann Ernte Zellen durch Trypsin mit 1 ml Trypsin-Lösung / Platte durch Neutralisation mit 10 ml von Dulbeccos modifiziertem Eagle-Medium (DMEM) gefolgt, die 2% Fetal Rinderserum (FBS) und 1% Penicillin / Streptomycin / Platte. </li><li> Übertragen suspendierten Zellen auf eine 50 ml konischen Röhrchen und zentrifugieren bei ~ 130 xg für 5 min, entfernen Überstandes und Resuspendieren Zellpellet wurde in 10 ml DMEM / 2% FBS / 1% Penicillin / Streptomycin. </li><li> Zählen Sie die Anzahl der Zellen mit einer Zelle Zähler, und dann eine Zell-Lösung mit 10 5 Zellen / ml, 150 ml halten Zellsuspension für den nächsten Schritt. Teller 10 4 Zellen (100 ul) in jede Vertiefung einer 96-Well-Zellkulturplatte mit einer Mehrpunkt automatisierten Liquid Dispenser (fortan Multidrop). Die Zellsuspension sollte ausreichend sein, in diesem Fall für die Beschichtung 12 x 96-Well-Platten. </li><li> Die Inkubation mit Zellen bei 37 ° C in einem Inkubator mit 5% CO 2 während der Vorbereitung der Transfektionsansatz. </li></ol> 2. Vorbereiten der Reporter Construct Stammlösung <ol><li> Isolieren hochwertige Reporterkonstrukt DNA unter Verwendung von Standard Midiprep Kits (NucleoBond Xtra Midi produziert vonClontech) und löst die DNA in einer Endkonzentration von 1 mg / ml für jede Reporter. </li><li> Bereiten Sie 100 ul Reporterkonstrukt Stammlösung durch die Kombination von 30 ul p53-FL, 30 ul 8X TCF-RL, 30 ul Elk1-Gal4, UAS 6 ul-CL und 4 ul H 2 O, um eine DNA-Mix mit erreichen eine endgültige 1:1:1:0.2 Verhältnis der Reporter. Endgültige DNA Konzentration dieser DNA-Mix stock Lösung beträgt 0,3 mg / ml. </li></ol> 3. Vorbereiten der siRNA / DNA Transfektion Mix In diesem Teil des Protokolls, bereiten wir siRNA / DNA Transfektion Mischungen, die ausreichen, um das Transfizieren 12 x 96-Well-Platten werden. Für diesen Test Bibliothek der 4 x 96-Well-Platte von siRNAs, werden wir jede siRNA Pool in dreifacher Ausfertigung transfektieren. Das Transfektionsreagenz wir verwenden heißt Effectene (Qiagen). In unseren Händen ist dies die einzige Reagenz, das für die gleichzeitige Aufnahme von siRNA und DNA in kultivierte Zellen funktioniert. <ol><li> Verdünnen Sie 80 ul Reporter Konstrukt stock solution in 8 ml EC-Puffer (Effectene Kit), um eine DNA-Lösung mit einer Endkonzentration von 3 pg / ml zu erreichen. In der Regel bereiten genug DNA-Lösung Mix für den sofortigen Einsatz. </li><li> Die Platte 20 ul dieser DNA-Lösung in jede Vertiefung einer 96-Well / Platte. Die Zahl der 96-Well-Platten auf, wie viele siRNA Pools getestet werden abhängen. Zum Beispiel kann in diesem Fall müssen 4 x 96-Well-Platten zu 384 verschiedene Pools siRNA bewerten. </li><li> Die siRNAs getestet werden sollte an einer Börse Konzentration von 5 &amp;mgr; m betragen. Wenn nicht, können sie replikaplattiert und verdünnt dieser Konzentration mit der empfohlenen Verdünnungspuffer mit jeder Bibliothek zugeordnet sind. </li><li> Übertragen 2 ul jeder 5 uM siRNA Lager in die entsprechende Vertiefung der PCR-Platte mit der verdünnten DNA Aktien mit einem Biomek Automated Liquid Handler. Je nach Umfang der Studie kann eine Mehrkanalpipette ausreichen. </li><li> Jetzt werden wir 1 ul Enhancer-Lösung (Effectene Kit) in jede Vertiefung der DNA / siRNA pla hinzufügente. Dies wiederum kann entweder mit einem automatisierten Liquid-Handling-oder Mehrkanal-Pipette erreicht werden. </li><li> Lassen Sie die Enhancer Reaktion bei RT für 5 min auftreten fügen Sie dann 3 ul Effectene Transfektionsreagenz in jede Vertiefung und Inkubation bei RT für weitere 10 min. </li><li> Zur Transfektion Komplexbildung zu stoppen, mit 10 ul DMEM / 2% FBS / 1% Penicillin / Streptomycin in jedes Well der PCR-Platte. </li><li> Werden 10 ul der Transfektionsgemisch in jede Vertiefung der 96-Well-Platte, welche die Zellen (wird aus der Zelle Inkubator). Da jeder Transfektion in dreifacher Ausführung durchgeführt werden, wird die gleiche Mischung zu zwei zusätzliche 96-Well-Platten mit Zellen eingesetzt werden. </li><li> Inkubieren der Zellen mit der Transfektion Mischungen bei 37 ° C in einer feuchten Umgebung mit 5% CO 2 für 36 Stunden zugegeben. </li></ol> 4. Bestimmen Luciferaseaktivitäten von Zellproben Nach 36 Stunden sind Luciferaseaktivitäten zur Messung bereitin transfizierten Zellen. Der Endpunkt sollte auf einer Pro-Experiment festgelegt werden. In unserer Studie war eine 36 h Inkubationszeit zuvor eine ausreichend robuste Signal nach dem Sinn Ergebnis Bestimmung ergab. Wie diese Studie umfasst verschiedene Reporter (ein in das Kulturmedium, und die beiden anderen im Zytoplasma der Zelle exprimiert sekretiert), wir Messungen sowohl Kulturmedium sowie ein Zelllysat zu erhalten. <ol><li> Nachweis von CL-Aktivität im Kulturmedium: <ol><li> 20 ul Kulturmedium replica-überzogen in einer weißen, undurchsichtigen 96-Well-Platte (entweder über die Biomek Liquid Handler oder Mehrkanal-Pipette). </li><li> In 20 ul CL-Assay-Puffer (Targeting Systems) in jede Vertiefung. </li><li> In 10 ul Cypridina Luciferinsubstrat (Targeting Systems) in jede Vertiefung und erkennen CL-Aktivität mit einem Luminometer (die Platte Pherastar Leser von BMG zum Beispiel produziert). </li></ol></li><li> Nachweis von FL und RL-Aktivität: <ol><li> Lyse der cEllen indem zunächst das Kulturmedium. Dies kann schnell durch Drehen Platte auf den Kopf und warf Medium in einer Senke erreicht werden. Verbleibende Medium kann durch leichtes Antippen der Platte kopfüber auf Küchenpapier entfernt werden. In 30 ul 1X Passive Lysis Buffer (Promega) in jede Vertiefung der Zellen unter Verwendung des Multidrop und Ort auf einer Plattform Wippe (Bellco ist ein Unternehmen, das diese macht) gesetzt eine mittlere Geschwindigkeit für 5 min bei RT. </li><li> In 20 ul des Luciferase Assay Reagent II (Teil des Dual-Luciferase Promega Kit) mit dem Multidrop und sofort messen FL Aktivität mit dem Luminometer. Als Nächstes fügen Stop &amp; Glo Reagenz (Promega), die die FL Aktivität auslöschen und erlauben die Messung von RL Aktivität. Hinweis: wenn Sie mit Substrat Mixe, die verlängerte Dauer des Signals (wie Dual-Glo Luciferase Kits von Promega) ermöglicht werden, erfordert die Verwendung von Flash-Kits schnelle Messung auf dem Substrat zusätzlich (innerhalb von 5 min). </li></ol></li></ol>

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	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. 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=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, B., 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=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, W., 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+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. 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OK und LL sind Autoren auf einen anhängigen Patent für Multiplex-Luciferase-Technologie.

Es gibt mehrere Lieferanten von Luciferase Reporter-basierte Systeme (wie Promega, Zielsysteme, New England Biolabs, und Thermo Fisher), die nützliche Online-Ressourcen zur Verfügung für diejenigen unterhaltsam ein Hochdurchsatz-Screening für die erste Zeit. Darüber hinaus sollte eine Reihe von ausgezeichneten Bewertungen in Bezug auf die Optimierung der Nutzung von High-Throughput-Screens für die Gen-Entdeckung und wie RNAi-basierte Screening-Ergebnisse mit anderen-omics-basierten Datensätzen integriert werden h...

Eine Vielzahl von Luciferase-basierte Reporter zur Überwachung einer Vielfalt von zellbiologischen Prozesse sind im Handel erhältlich. Die Mehrzahl dieser DNA-Konstrukte kodieren Proteine, die Luciferase induziert durch spezifische Reize Zelle oder Störungen auszudrücken. Die robuste transkriptionell basierte Reportern platzieren die Expression eines Enzyms Luciferase unter der Kontrolle von synthetischen Enhancer-Elemente oder Gen-Promotor-Regionen für ihre Zuverlässigkeit in der Berichterstattung eine physiologisch relevante Transkriptions-Veranstaltung 1,2 etabliert. Es gibt auch trans-Reporter Luciferase-Systeme, die GAL4-UAS zu nutzen, um Luciferase Proteinexpression fahren. Gal4 ist ein Hefe-Transkriptions-Aktivator und die UAS ist ein Enhancer, die Gal4 spezifisch bindet, um die Transkription der Gensequenzen angeordnet stromabwärts davon 3 zu aktivieren. Diese Arten von Luciferase-Systeme sind in der Regel von zwei DNA-Plasmide getragen - ein GAL4-Protein kodiert, das fusioniert an die regulatorischery Teil eines Proteins von Interesse, und die zweite Luciferase kodiert für ein Protein unter der Kontrolle eines oder mehrerer UAS-Sequenzen angeordnet. Somit spiegelt die zelluläre Luciferasesignal die Aktivität des Proteins von Interesse. Eine andere übliche Anwendung von Luciferase-basierte Reporter dient zur Überwachung Proteinstabilität, wodurch ein Protein von Interesse an einer Luciferaseenzyms 4 fusioniert ist. Unabhängig von der Art der Reporter wird ein Gewährleistungsausschluss hier kann man nicht davon ausgehen, die Spezifität von jedem Reporter zu gut verhört haben zur Kenntnis genommen. Somit wird Due Diligence in den Einbau von neuen Reporter-Konstrukte in jeder Forschung Strategie erforderlich. Die Auswahl des Luciferaseenzyms Auslesen kann ebenso wichtig sein wie die Zuverlässigkeit der DNA-Elemente, die ihren Ausdruck kontrollieren. Während Glühwürmchen-Luciferase (FL) ist das am häufigsten verwendete Enzym in im Handel erhältlichen Konstrukte wird das Aufkommen der neuen Luciferase-Reporter-Systeme, die keine ATP (wie im Fallvon FL-basierte Reaktionen) oder dass integrieren stabiler Enzyme, die stärkere Signale aussenden versprechen, um die Effizienz und Zuverlässigkeit dieser Forschungsplattform zu verbessern. Ein wichtiger Hinweis für die Auswahl der Luciferase Reporter in chemische Untersuchungen ist die Anfälligkeit von FL auf chemische Hemmung, die Anlass zu falschen Berichten geben könnte. Nach unserer Erfahrung sind auch andere Enzyme wie Renilla Luciferase oder Gaussia (RL und GL, respectively), die Coelenterazin verwenden als Substrat weniger leicht gehemmt durch kleine Moleküle 5. Das gängigste Format zum Multiplexen Luciferase Auslesen bringt die Verwendung von FL und RL weitgehend angesichts der Verfügbarkeit von easy-to-use-Kits mit der Fähigkeit, nacheinander messen enzymatischen Aktivitäten aus einer einzigen Probe. In den meisten Kits, werden die Proben zunächst auf Luciferin ausgesetzt Ebenen der FL Aktivität durch eine Quenchreagens gefolgt, die gleichzeitig mit Coelenteracin abgeschieden wird, um eine gebraucht ergeben offenbarendary RL Signal. Mit dem Zusatz von anderen Luciferasen, wie GL oder dass auf ein weiteres Substrat, wie Cypridina Luciferase (CL), eine größere Anzahl von Möglichkeiten zur Erzeugung von Daten mit hohen Luciferasen entstanden abgesondert werden kann. Ein Beispiel für eine genomweite RNAi-Bildschirm mit Hilfe dieser Technik können hier 6 gefunden werden. Diese technologischen Fortschritte haben wiederum trieb die Entwicklung von spezifischen Mechanismen, um jede Art von Luciferaseenzyms stillen, um cross-talk 7 begrenzen. In unseren Händen, stellen wir eine größere Häufigkeit von &quot;Randeffekte&quot; (unerklärliche Trends in Zellaktivität mit der Kante des hohen Titer Kultur Platten verbunden sind), mit abgesondert Luziferasen wie GL oder CL. Auf der anderen Seite sind solche sezerniert Luciferasen aus kinetischen Assays nützlich, da sie Signalabtastung ohne Verfälschung Zelllebensfähigkeit ermöglichen. Für unsere hier vorgestellten Studie, werden wir gleichzeitig die Aktivitäten der drei Krebs-relevantezelluläre Prozesse: die p53, Kras und Wnt Signaltransduktion. Die Reporter in unserer Studie einbezogen sind die pp53-TA-Luc FL Reporter von Clontech (im Folgenden auch als p53-FL Reporter bezeichnet) 8, eine Elk1-GAL4/UAS-CL Reporter System (im Folgenden Elk-1-Reporter, Agilent), und die 8X TCF RL Reporter, die mehrere synthetische Enhancer-Elemente von den Wnt-Signalweg Transkriptionsfaktoren wie T-Zell-Faktoren (oder TCFs) 9-11 bekannt anerkannt enthält.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

Genome-scale Verhör der Genfunktion mittels RNA-Interferenz (RNAi) birgt ein enormes Versprechen für die schnelle Identifizierung von chemisch gefügig Krebszelle Schwachstellen. Die Begrenzung der Potenzial dieser Technologie ist die Unfähigkeit, schnell zu beschreiben die mechanistische Grundlage der Ergebnisse und somit phänotypische informieren die Entwicklung von molekular zielgerichtete therapeutische Strategien. Wir skizzieren hier Methoden zur zellulären Phänotypen durch RNAi-vermittelte Gen-Targeting mit Multiplex-Reporter-Systeme, die Überwachung der wichtigsten Krebszelle-assoziierte Prozesse ermöglichen induziert dekonstruieren. Diese High-Content-Screening-Methode ist vielseitig und kann leicht für das Screening von anderen Arten von großen molekularen Bibliotheken angepasst werden.

Trotz der Fortschritte bei der Kartierung der Mutations-Landschaft von verschiedenen Krebsarten mit massiven Genom-Sequenzierung Bemühungen 12-14, haben wir noch nicht im Ort haben einen systematischen Ansatz für die Umsetzung dieser Beobachtungen in Interventionsstrategien. In Darmkrebs (CRC), Mutationen, die voraussichtlich drei Komponenten des zellulären Prozesse beeinflussen werden - Wnt / β-Catenin, p53 und Kras-Signalisierung - sind in 99% aller Tumoren hindeutet, sie sind wichtige Treiber der zellu...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Das Erreichen einer Systemebene Verständnis zellulärer Prozesse ist ein Ziel der modernen Zellbiologie. Wir beschreiben hier Strategien zum Multiplexen Luciferase Reporter von verschiedenen zellulären Funktion Endpunkte zur Genfunktion mit genomweiten RNAi-Bibliotheken zu verhören.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

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11.0K Aufrufe.  UT Southwestern Medical Center. Das Erreichen einer Systemebene Verständnis zellulärer Prozesse ist ein Ziel der modernen Zellbiologie. Wir beschreiben hier Strategien zum Multiplexen Luciferase Reporter von verschiedenen zellulären Funktion Endpunkte zur Genfunktion mit genomweiten RNAi-Bibliotheken zu verhören.

Serie: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Wie kortikale Faltung von MR-Bildern messen: eine Schritt-für-Schritt-Anleitung zum Local Gyrification Index Compute

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Forschung

Medizin

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

Die Verwendung von Zystometrie in kleine Nagetiere: A Study of Bladder Chemosensorik

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening für Melanom Modifiers mit einem Zebrafisch Autochthone Tumormodell

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Ein hoher Gehalt<em&gt; In Vitro</em&gt; Pancreatic Islet β-Zell-Replikation-Discovery-Plattform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Screening bioaktiver Nanopartikel in phagozytischen Immunzellen für Inhibitoren der Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Routine-Screening Methode für Mikropartikel in Thrombozytentransfusionen

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Hydra, eine Computer-basierte Plattform für die Unterstützung der Kliniker in der Herz-Kreislauf-Analyse und Diagnose

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In-vitro- Methode zur Kontrolle Konzentrationen Halogenkohlenwasserstoffe in kultivierten alveolären Epithelzellen

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...