We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Tier Arbeit an der USGS National Wildlife Health Center durchgeführt wurde, in Übereinstimmung mit der NIH Office of Laboratory Animal Welfare Richtlinien und unter institutionellen Tierpflege und Verwendung Ausschussprotokoll # EP080716 geführt. Gewebe von Jäger und geerntet Tiere waren Geschenke von der Wisconsin und Michigan Abteilungen für Naturressourcen. 1. Herstellung der Lösung HINWEIS: Die unten aufgeführten Lösungen sind für die Vorbereitung der CER-Assay-Substrat in Abschnitt 2 unten erforderlich. Planen jeder dieser Lösungen von höherer Konzentration Stammlösungen; empfohlene Konzentration für Stammlösungen werden in Klammern nach jeder jeweiligen chemischen Bezeichnung aufgeführt erhalten. Bereiten Sie alle Lösungen frisch auf Untergrundvorbereitung und bei 4 ° C bis zur Verwendung. <ol><li> Für Lysepuffer, bereiten eine Lösung der folgenden in 10 mM Tris, pH 7,5 (1 M Tris, pH 7,5, Stammlösung empfohlen): 100 mM sOdium Chlorid (NaCl, 1 M Stammlösung), 10 mM Ethylendiamintetraessigsäure (EDTA, 500 mM EDTA, pH 8,0 Stammlösung), 0,5% NP-40 (10% Stammlösung), 0,5% Desoxycholat (DOC, 10% Stammlösung ). </li><li> Zur Umwandlung Puffer wird eine Lösung aus 0,05% Natriumdodecylsulfat (SDS, 10% ige Stammlösung) und 0,5% Triton X-100 (10% -ige Stammlösung) in 1 phosphatgepufferter Salzlösung (PBS, 10, pH 7,4 Stammlösung). </li><li> Für chaotropen Lösungen, bereiten zwei 3 M Lösungen von Guanidinhydrochlorid (GdnHCl, 8 M Lager) in 1x PBS (10x Stammlösung). Der pH-Wert einer der Lösungen auf 3,5 ± 0,05 unter Verwendung von konzentrierter Salzsäure (HCl). Prüfen, ob der pH-Wert der zweiten Lösung bei pH 7,4 ± 0,05 bleibt und stellen mit konzentrierter HCl oder Natriumhydroxid (NaOH), falls erforderlich. </li></ol> 2. Bereiten Sie CER Assay Substrat-Paare <ol><li> Erhalten gesunde, nicht-TSE-infizierten Hirngewebe von Spezies von Interesse und bereiten eine 10% Gewicht pro Volumen(W / v) Homogenat in Lysepuffer mit einer der folgenden Methoden: Dounce, Kugelmühle oder Mörser und Stößel Homogenisierung. Weiter verfeinern resultierenden Homogenate indem sie einer Spritze und Nadel-Homogenisierung mit Nadeln zunehmender Spur bis Substrat abgesaugt und mit Leichtigkeit durch eine 27G-Spritzennadel ausgestoßen werden. <ol><li> Für Substrate aus Nagetiere und andere kleine Säugetiere hergestellt homogenisieren gesamte Gehirn (s); für Substrate von größeren Säugetieren bereit, verwenden Obex (Hirnstamm) Gewebe-Homogenate vorzubereiten. Bei der Wahl der Menge von Hirnhomogenat vorzubereiten nicht mehr als 50% der Kapazität der Zentrifuge zur Eiweißfällung in Schritt 2.6 verwendet. </li><li> Wenn die Qualität der erworbenen Hirngewebe ist fraglich, bewerten PrP-C-Spiegel durch SDS-PAGE und Immunoblot 16 17 vor der Untergrundvorbereitung. </li></ol></li><li> Teilen Sie Hirnhomogenat in zwei gleiche Volumina in separaten gekennzeichnete Kunststoff konische Röhrchen. Fügen GdnHCl (3 M Lösung in 1 PBS) bei entweder pH 3,5 oder 7,4 zu jedem Röhrchen in einem 1: 1 Volumenverhältnis zu Hirnhomogenisat. </li><li> Überprüfen Sie die pH-Werte der beiden Lösungen. Den pH-Wert der pH 3,5 Substrat auf pH ± 0,05 unter Verwendung von konzentrierter HCl. Sicherstellen, dass der pH-Wert des anderen Substrats bei pH 7,4 ± 0,05 bleibt und der pH wird, falls notwendig, mit HCl oder NaOH nach Bedarf. Vermeiden Schwingen pH-Werte durch sorgfältige Zugabe von Säure oder Base. </li><li> Drehen Lösungen auf einem End-Over-End-Mixer bei Raumtemperatur (RT) für 5 Stunden. </li><li> Auszufällen Protein durch Zugabe vier Volumina Methanol zu den Substratlösungen in jeden konischen Rohr, Wirbel um gut zu mischen, und Inkubation bei -20 ° C für 16-18 Std. </li><li> Sedimentproben durch Zentrifugation bei 13.000 × g für 30 min bei 4 ° C. Dekantieren Sie vorsichtig und entsorgen Sie die Überstände und lassen Methanol aus den Pellets verdampfen. Verwenden Sie Wattestäbchen, um bei der Aufnahme von Methanol zu der Innenseite des Rohres klammerte unterstützen. Lassen Sie keine Pellets zu über-dry und einmal Methanol nicht mehr sichtbar ist, mit dem nächsten Schritt fortzufahren. </li><li> Resuspendieren Proteinpellets in Konvertierungspuffer. Verwenden einer Menge des Umwandlungspuffers gleich der Menge Hirnhomogenat Ausgangsmaterial in Schritt 2.2 in jedem Röhrchen verteilt. HINWEIS: Es ist wichtig, dass die Umwandlung Puffer verwendet werden, um Protein-Pellets von beiden pH 3,5 und 7,4 behandelte Substrat Vorbereitungen resuspendieren wird die in Schritt 1.2 (die geringe Reinigungsmittel und einen physiologischen pH-Wert enthält) definiert Lösung. </li><li> Kurz beschallen Substratlösungen in einem cuphorn Ultraschallgerät (10 sec bei ~ 30% der maximalen Leistung), Aliquots in 0,5 bis 1,0 ml-Volumina in der Bezeichnung 1,5-ml-Mikrozentrifugenröhrchen. Führen Sie eine Qualitätskontrolle Immunoblot (Schritt 2.9) aus einem aliquoten Teil von jedem Substrat. Bewahren anderen Aliquots bei -80 ° C bis zum Gebrauch CER-Test (Abschnitt 4) durchzuführen. </li><li> Führen Sie die Qualitätskontrolle auf CER Substrat-Paare vor der Anwendung (Abbildung 2). Diese Schritte sicherzustellen, dass CER Substrate wird provide optimale Ergebnisse bei der Umwandlung Studien. <ol><li> Stellen Sie sicher, vergleichbare Mengen an PrP C in den beiden Substraten. Vergleichen PrP Ebenen in 10-25 ul jedes Substrat durch SDS-PAGE und Immunoblotting 16 17. Wenn Protein-Spiegel in der pH 7,4 und 3,5 Substrate innerhalb von ~ 10% voneinander unterscheiden, sind die Substrat-Paare für die Verwendung in CER Studien geeignet. HINWEIS: Die PrP Immunoblots nicht Beweise für umfangreiche PrP C Abbau zeigen. Das PrP Immunreaktivität sollte vor allem&gt; 20 kDa ist. Bands geringer als dieser Molekülmasse können Abbauprodukte sein. Bandenmuster sollte etwa gleich zwischen Substrat-Paare sein. </li><li> Da die PrP C in beiden Substraten sollte PK empfindlich sein, behandeln 10-25 ul jedes Substrats mit PK in einer Endkonzentration von 100 ug / ml und verdauen Proben bei 1000 Upm bei 37 ° C für 1 Stunde in der Thermoschüttler. Beurteilen verbleibende PrP Signals durch SDS-PAGE und Immunoblotting 16 17.Substrate mit PrP res sind nicht für den Einsatz in CER Studien geeignet. </li></ol></li></ol> 3. Bereiten Sie TSE-Agent Seeds <ol><li> Erhalten TSE-infizierten Hirngewebe von Spezies von Interesse und bereiten eine 10% (w / v) Homogenat in 1x PBS, pH 7,4, unter Verwendung der in Schritt 2.1 oben genannten Methoden. HINWEIS: Saatgut kann auch aus anderen TSE-infizierten Geweben außerhalb des zentralen Nervensystems hergestellt werden, aber die Effizienz der Umwandlung der aus dem Gehirn stamm Substraten durch Samen von TSE-infizierten peripheren Geweben abgeleitet wurde noch nicht experimentell in der Literatur untersucht. </li><li> Aliquoten resultierende Homogenat (n) in 100-300 ul-Volumina in 0,5 ml Mikrozentrifugenröhrchen und bei -80 ° C, bis bereit, die CER-Test durchzuführen. </li></ol> 4. CER Assay HINWEIS: Die CER Test umfasst Zugabe einer relativ kleinen Menge von PrP TSE (in der &quot;Samen&quot; zur Verfügung gestellt) von einer Speziesin einen relativen Überschuss von PrP C von einer anderen Spezies, das teilweise an entweder pH 3,5 oder 7,4 denaturiert (in der nicht-infizierten Hirn &quot;Substrat&quot;, zur Verfügung gestellt). Nach einer längeren Schütteln Inkubationszeit wird vorlagengesteuerten Umwandlung von PrP C von PrP TSE in jeder Reaktion durch densitometrische Signal des PrP res übrigen nach PK-Verdau und Immunoblot-Nachweis untersucht. Vergleichen PrP res Ebenen der Substrate zuvor bei pH 3,5 vs. 7,4 denaturiert liefert ein Maß für die Artenbarriere. Eine experimentelle Übersicht dieses Testverfahrens ist in 1 bereitgestellt. <ol><li> Langsam tauen nicht infizierte CER Substrat-Paare (Assay erfordert gleiche Volumina Substrate zuvor sowohl auf pH 3,5 und 7,4 denaturiert) und TSE-infizierte Saatgut-Lösungen, indem sie auf einem Bett aus Eis (ca. 1 Stunde auftauen Zeit). </li><li> Sobald sie vollständig aufgetaut, absaugen und dann vertreiben jedes Substratlösung durch eine27 G Injektionsnadel mehrmals neu zu homogenisieren. Kurz beschallen jeden TSE-infizierten Samen Lösung für 10 s bei ca. 30% der maximalen Leistung. Bewahren Sie alle Lösungen auf Eis während der Vorbereitung Umwandlungsreaktionen. </li><li> Label 5 Einzel geringe Bindungs, dünnwandigen PCR-Röhrchen pro TSE-Erreger getestet. </li><li> Bereiten Umwandlungsreaktionen in diesen Rohren durch Zugabe von 5 ul 10% TSE-infizierten Samen Lösung in 95 &amp; mgr; l von (a) CER Substrat bei pH 7,4 und (b) CER Substrat bei pH 3,5 hergestellt. <ol><li> Für jede Versuchsprobe, bereiten Parallelreaktionen in CER Substrat-Paare zuvor sowohl auf pH 3,5 und 7,4 denaturiert. </li><li> Optimieren Verhältnisse von TSE-Erregern Samen nicht infizierten Gehirnsubstrat durch empirische Bestimmung. Gemeinsame Samen: Substrat Verhältnissen eingesetzt pflegen eine Gesamtreaktionsvolumen von 100 ul und beinhalten 1:99 ul, 2:98 und 5:95 ul ul. Für die meisten Anwendungen, die 5:95 ul Samen: angemessen ist Substrat-Volumen-Verhältnis und ist das, was in th vorgestelltist Protokoll. </li></ol></li><li> Bereiten Sie drei Kontrollproben pro TSE-Erreger wird wie folgt geprüft: (a) mit 5 ul 10% TSE-infizierte Saatgut Lösung auf 95 &amp; mgr; Konvertierungspuffer als Kontrolle für Eingabe PrP res; mit 5 ul Konvertierungspuffer 95 &amp; mgr; l Substrat in (b) pH 3,5 und (c) pH 7,4 als Kontrollen für nicht-spezifische, nicht-gestützten Umwandlung vorbereitet. </li><li> Vortexen Sie jede Probe in seiner geschlossenen PCR-Röhrchen, um Saatgut und Substrat gut mischen. Folgen Sie mit einem momentanen Impuls in einem Low-Speed-Tisch Mini-Zentrifuge, jede Volumen des Reaktionsgemisches in der PCR-Röhrchen Deckel gefangen zu entfernen. </li><li> Tragen Sie die Proben in individuell-markierten PCR-Röhrchen in einer Tischthermoschüttler ausgestattet, um PCR-Röhrchen zu akzeptieren. Schütteln Proben bei 1000 Upm bei 37 ° C für 24 Stunden. </li><li> Nach Umschütteln Sarcosyl in einer Endkonzentration von 2% (w / v) und PK auf eine Endkonzentration von 100 ug / ml. Digest Proben bei 1000 Upm bei 37 ° C für 1 Stunde in der Thermoschüttler. NHINWEIS: Die Zugabe von Sarkosyl Proben nach der Konvertierung hilft bei der PK Verdauung von PrP C. Falsch positive Signale in ungeimpften Proben (Schritt 4.5) praktisch durch Zugabe von Sarkosyl eliminiert. </li><li> Hinzufügen SDS-PAGE-Probenpuffer, Wärme Proben auf 95 ° C für 5 Minuten und Lösung verbleibenden PrP res in jeder Probe durch SDS-PAGE 16 gefolgt von Immunoblotting 17. HINWEIS: Nach Zugabe von Probenpuffer und Heizung, Proben können sofort verarbeitet werden oder Proben können bei -20 ° C oder vor dem Immunoblotting gespeichert -80ºC. Alle hier vorgestellten Daten wurden unter Verwendung der Novex NuPAGE Gel und Transfersysteme erzeugt. Einzelne Proben aus dem CER-Test wurden mit Probenpuffer und Reduktionsmittel nach Herstellerangaben behandelt. Die Proben wurden anschließend bei 95 ° C für 5 min erhitzt und 30 ul jeder Probe wurde in eine 12% Bis-Tris Fertiggelen geladen. </li><li> Berechnen des Umwandlungswirkungsgrad (CER) als MaßArtenschranke. Maßnahme Ebenen PrP res densitometrisch 17 und unterteilen die Gesamtdichte des pH 7,4 Probe durch diejenige des pH 3,5 Probe. Mit 100 multipliziert, um einen Prozentsatz zu produzieren. </li><li> Kompilieren von Daten aus unabhängigen experimentellen Wiederholungen, um eine mittlere CER ± Standardabweichung für eine Spezies mit einer bestimmten TSE-Erreger zu generieren. </li></ol>

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The authors have nothing to disclose.

Bei erfolgreichem Abschluss des Protokolls sollte die Aufmerksamkeit auf PrP-C-Spiegel bei nichtinfizierten zur Untergrundvorbereitung (Schritt 2.1.2) und der Samen-Verhältnis für Umwandlungsreaktionen (Schritt 4.4.2) Substrat verwendet Hirngewebe zu zahlen. Nach unserer Erfahrung können Gehirne für die Verwendung als CER-Substrat nach einem beträchtlichen Zeitraum post mortem entnommen werden, solange PrP C durch Immunoblotting (Abbildung 4) vorhanden ist. In der Tat wurden e...

Transmissible spongiforme Enzephalopathien (TSE, Prionenerkrankungen) sind eine Gruppe von tödlichen neurodegenerativen Erkrankungen mit verlängerten Inkubationszeiten, die eine Vielzahl von Tieren und Menschen zu beeinflussen. Die mutmaßliche ätiologische Agens von TSE besteht aus einer fehlgefalteten Isomer der Host Prionprotein (PrP), die in der Lage zur Selbstausbreitung von vorlagengesteuerten Umwandlung der normalen zellulären Form PrP (PrP C) in ein infektiöses, krankheitsassoziierte ist umfasste Formular (PrP TSE), die im zentralen Nervensystem Gewebe des infizierten Wirts 1 ansammelt. Infektiöse Prionen übertragen Säugetier Allgemeinen von Host-zu-Host in einem artenspezifisch, was zu dem Begriff der &quot;TSE Artengrenze&quot; Begrenzung Wirtsartwechsels Ereignisse 2 gegeben hat. Die biologischen Determinanten des Prion-Spezies-Barriere sind nicht gut verstanden. Aminosäuresequenz-Ähnlichkeit zwischen der infektiösen PrP TSE und dem Host-PrP C cein stark beeinflussen, ob die Umwandlung erfolgt 3-5, bleibt aber nicht aus, um alle in vivo 6,7 beobachtet Prionenübertragung Ereignisse erklären. Somit hat die Charakterisierung von TSE Artschranken weitgehend von Tierexpositionsstudien herangezogen: Belichten naiven Tieren aus einer bestimmten Art von Prionen von einer anderen und durch Messen der resultierenden Inkubationszeit zu Beginn der Erkrankung und Erkrankungsrate als Indikator für die Übertragungseffizienz. Mäuse, die PrP C von heterologen Spezies werden auch für diese Art von Untersuchung 8 verwendet. Die Kosten im Zusammenhang mit transgenen Maus Produktion und langwierigen Prion Biotests sowie ethischen Überlegungen der Tiernutzung verbunden sind, sind Hindernisse für die experimentelle Untersuchung von TSE Artgrenzen. Bewertung der menschlichen Spezies-Barriere auf TSE vertraut auf Mäuse entwickelt, um menschliche PrP C auszudrücken. Diese Mäuse erfordern langen Inkubationszeiten, um die menschliche TSE oder Modifikationen an t erliegener menschlichen PrP-C-Molekül für eine schnelle Krankheitsbeginn 9. Inkubationszeiten für nicht-menschlichen TSE bei diesen Mäusen kann über die normale Maus Lebensdauer machen Interpretation der negativen Ergebnissen anspruchs erweitern. Nichtmenschliche Primaten sind auch als Stellvertreter für das Studium der menschlichen Spezies Barrieren verwendet worden, aber diese Studien behaftet mit den gleichen Herausforderungen wie andere Arten von Tierversuchen und nicht-menschlichen Primaten nicht genau rekapitulieren Krankheit, wie es in den menschlichen Wirt geht. Tier Biotests bleiben der &quot;Goldstandard&quot; Verfahren zur Messung der Anfälligkeit einer Spezies auf eine TSE, aber die Hürden, Kosten und Ethik dieser Live-Tierstudien Untersuchung Alternativen gezwungen. Eine Reihe von in vitro-Tests, basierend auf der Beurteilung der Umwandlung von Wirts PrP C zu einer Proteinase K (PK) beständigen Zustand (PrP res), wenn sie von PrP TSE ausgesät, wurden entwickelt und verwendet, um zu untersuchen TSE spECIES Barrieren 10-12. Beispiele für in-vitro-Tests sind zellfreie Umwandlung Assays PMCA (PMCA) und die Umwandlungseffizienz-Verhältnis (CER) Assay 10-14. Während keiner dieser Tests in Betracht periphere Faktoren Artgrenzen nach einer natürlichen Infektion beteiligt zu nehmen, kann sinnvoll sein, alle potenziell anfälligen Wirten für TSE zu identifizieren. Hier präsentieren wir das Protokoll für die CER-Test, bei dem zwei denaturiert PrP C Substrate von normalen Hirnhomogenaten abgeleitet sind in Tisch Prion Umwandlungsreaktionen verwendet (Abbildung 1) 13. PrP C in der bei pH 7,4 denaturiert Substrat kann nur an PrP res von PrP TSE umgewandelt werden ausgesät in die Umsetzung in Abwesenheit eines Speziesbarriere 14. Demgegenüber Denaturierung von PrP C in dem anderen Substrat bei pH 3,5 erlaubt es, an PrP res nach Inkubation umgewandelt werdenmit PrP TSE von irgendeiner Spezies und dient als Kontrolle für die Konvertierung. Das Verhältnis der Umwandlung von PrP C zu PrP res in pH 7,4 Substrats relativ gegenüber der pH 3,5 Substrat liefert ein Maß für die Artenbarriere. Wir haben festgestellt, dass die CER-Test prognostiziert bekannten Arten Barrieren der Labormäuse, verschiedene TSE und haben den Test bei den Bemühungen um die Artengrenze zahlreicher Säugetierarten, einschließlich Dickhornschafe, Chronic Wasting Disease (CWD) und anderen TSE 14 vorherzusagen, 15. Ermittler an einer Werkzeug ermöglicht ein schnelles Screening von TSE Artgrenzen oder Bewertung von PrP C to-PrP res Konvertierung wird diese Methode nützlich.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Der erfolgreiche Einsatz des CER Assay hängt weitgehend von der Qualität der in Umwandlungsreaktionen verwendeten Substrat-Paaren. Aus diesem Grund folgt vor, um CER Substratpaare vorbereiten, eine Qualitätskontrolle Immunoblot sollte (Abbildung 2) durchgeführt werden. Für beide Substrate Assay 10-25 ul durch Immunoblotting. PrP C sollte in jedem Substrat leicht nachweisbar sein und PrP C Ebenen müssen etwa gleich zwischen den beiden. Immunreaktivität wird vor allem sichtbar...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Die Beurteilung Transmissible Spongiforme Enzephalopathie Artgrenzen mit ein<em&gt; In-vitro-</em&gt; Prion Protein-Umwandlungsassay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K Aufrufe.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Serie: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Forschung

Medizin

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Die Beurteilung Teratogene Änderungen in einem Zebrafisch-Modell der Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

Isolierung von löslicher und unlöslicher PrP Oligomere in das normale menschliche Gehirn

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulation von Bauchspeicheldrüsenneuroplastizität:<em&gt; In-vitro-</em&gt; Zwei-Neuron-Plastizität Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Verwendung Adeno-assoziierte Virus als Werkzeug Retinal Barrieren in Disease Study

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Ein Ferkel Modell Neonatal hypoxischischämischer Enzephalopathie

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modellierung Enzephalopathie praematurorum Mit Prenatal Hypoxie-Ischämie mit Intra-Frucht Lipopolysaccharide bei Ratten

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Die Beurteilung Spezifität von Anti-Krebs-Medikamente<em&gt; In Vitro</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin-induzierte Quellung in Intestinale Organoide: Ein<em&gt; In Vitro</em&gt; Assay für Fibrosis Patienten in Cystic Drug Response-Beurteilung

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Neonatal Murine Cochlear Explant Technik als<em&gt; In vitro</em&gt; Screening-Tool in der Hörforschung

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...