Wir möchten Dr. Philippe Erbs, die uns mit der Reihenfolge der FCU1 Gen danken. Diese Arbeit wurde vom NIH AI 26649 unterstützt.

1. FCU Negative Selection System in Entamoeba histolytica <ol><li> Der Vektor Steuerleitung (HM1 / pKT3M) und die experimentelle Linie (HM1/pKT3M-FCU1) wurden von bisher beschriebenen Techniken 1,2 erzeugt. Seed die Linien unter-Studie aus dem Bestand Röhren in T-25 Flaschen und pflegen Auswahl durch Zugabe geeigneter Antibiotika (12 ug / mL G418) in TYI-S-33 3 Medium. Die Flaschen sollten 70-80% konfluent nach 16-18 Stunden Wachstum bei 37 ° C. </li><li> Bereiten Sie einen frischen 50 mM Stammlösung von 5-Fluorocytosin (5-FC, Sigma) in warmen TYI-S-33 Medium. Vortex konsequent für einige Minuten auf 5-FC vollständig aufzulösen. Filter-Sterilisation der Stammlösung, indem es durch ein 0,22 um-Filter. </li><li> Ernte Trophozoiten, indem die Flaschen auf Eis für 3-5 Minuten und das Sammeln der Zellen durch Zentrifugation bei 200xg für 5 Minuten bei Raumtemperatur. Das Pellet in frischem Medium TYI und zählen Sie die Zellen. </li><li> Aufbau eines typischen Experiments verwendet 0,5 x 10 4 Zellen pro Vertiefung einer 48-Well-Platte in einem Gesamtvolumen von 1 mL und dreifach wiederholt und für jede Prüfbedingung. Darüber hinaus erleichtert Fluoreszenz-Messungen, ist eine separate Platte für jeden Stamm pro Zeitpunkt verwendet. </li><li> Seed 0,5 x 10 4 Zellen pro Vertiefung für jeden Test 5-FC-Konzentration in dreifacher Ausfertigung. Pflegen Sie die Antibiotika-Selektion Druck in der TYI Medium. Nun fügen Sie die erforderliche Menge von 5-FC-Stammlösung und Trophozoiten in jede Vertiefung. </li><li> Die Inkubation in einer anaeroben Beutel bei 37 ° C. </li></ol> 2. Bestimmung der Auswahl Efficiency <ol><li> Bereiten Sie 2 mg / ml Stammlösung von Cell-Tracker grünen CMFDA (Invitrogen) in DMSO. Um die Arbeit zu verdünnen das Lager 1000-fach in serum-freiem Medium TYI. </li><li> Entfernen Sie das Medium vollständig von Trophozoiten Kulturplatten und ersetzen mit 150 ul serumfreiem TYI mit CMFDA. Weiter Inkubation bei 37 ° C für 1 Stunde. </li><li> Entfernen Sie die Farbstofflösung aus Brunnen und lesen Sie die Platte in der Spectramax M2 Mikroplatten Spektrofluorometer. Die Anregungswellenlänge sollte bei 492 nm eingestellt werden und die Fluoreszenzemission bei 517 nm gelesen. </li><li> Vergleichen Sie die Fluoreszenz-Werte für die Kontroll-Zellen im Vergleich zu den Test-Transfektanten. </li><li> Lesen Sie weiter die Platten bei jedem Zeitintervall. Verwenden Sie geeignete positive (TYI nur) und negativen (25 ug / mL Hygromycin) Kontrollvertiefungen in jeder Platte. </li></ol> 3. Repräsentative Ergebnisse Die E. histolytica Transfektanten zeigten robust Ausdruck Codon optimiert rekombinanten FCU1 (Abbildung 1). Wenn dieses Protokoll korrekt ist es Ergebnisse in selektiven Eliminierung von E. gefolgt histolytica exprimieren FCU1 in Gegenwart von 0,5 mM 5-Fluorocytosin. Control-Zellen, im Gegensatz normal weiter wachsen bis zu 5 mM Konzentration von 5-FC und erreichen Zusammenfluss zwischen 72 und 96 Uhr (Abb. 2-a). Die FCU1 tragenden Zellen in 48 Stunden vollständig lysiert, wenn die behandelte Brunnen unter einem Mikroskop betrachtet werden. Diese auch verringerte Fluoreszenz zeigen, wenn mit den vitalen Farbstoff CMFDA, die in quantitative Studien, bei denen große Anzahl von Proben (Abb. 2-b) verwendet wird gefärbt. Die FCU1/5-FC System ist ein effektives und leistungsfähiges Werkzeug für die negative Selektion E. histolytica Trophozoiten. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Generierung von E. histolytica HM1 Transfektanten, die negativen Selektionsmarker (A) FCU1 rekombinante Protein wurde auf einem Western-Blot mit anti-c Myc-Antikörper (oben) erkannt. Wie erwartet, konnte ein 42kDa-Band erkannt (dargestellt durch einen Pfeil) an der gesamten Lysat aus der FCU-Transfektanten, aber nicht in dem Vektor allein Steuerung Transfektanten. Der untere Teil zeigt den gleichen Blot sondiert mit anti-Aktin-Antikörper als Ladekontrolle. (B) Ergebnisse der quantitativen real time PCR zeigen Ausdruck FCU1 spezifische mRNA normiert auf lgl4 Kontrolle. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="Abbildung 2" /> Abbildung 2. In-vitro-Empfindlichkeit gegenüber Medikamenten von E. histolytica Transfektanten, die negativen Selektionsmarker Überlebenskurven von (a) Steuerung Transfektanten und (b) FCU1 Ausdruck Transfektanten in 48-Well-Platte kultiviert. Die Zellen wurden in Anwesenheit von steigenden Konzentrationen von gewachsenen das Pro-Pharmakon-5-Fluorocytosin; das Überleben der Zelle wurde durch CMFDA Färbung in jedem Zeitintervall quantifiziert werden, da auf der X-Achse bezeichnet. Die Y-Achse repräsentiert Fluoreszenz-Einheiten und ist ein direktes Spiegelbild der überlebenden Zellpopulation. Bitte beachten Sie, dass die FCU1 Ausdruck Transfektanten signifikante Sensibilität zeigte bei 0,5 mM Prodrug-Konzentration im Vergleich zur Kontrollgruppe. Keine Zellen wurden bei 120h Zeitpunkt in dieser Brunnen als com beobachtetVergleich zum konfluenten Wachstum in den entsprechenden Kontroll-Wells unter dem Mikroskop (Daten nicht gezeigt). Hygro bezeichnet 25 Mirkogramm / ml Hygromycin als negative Kontrolle verwendet.

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. Microbiol. 45, 656-660 (1998).</li><li>Erbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cancer+gene+therapy+by+adenovirus-mediated+transfer+of+a+bifunctional+yeast+cytosine+deaminase/uracil+phosphoribosyltransferase+fusion+gene.">In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.</a> Cancer Res. 60, 3813-3822 (2000).</li><li>Braks, J. A. M., Franke-Fayard, B., Kroeze, H., Janse, C. J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+positive-negative+selectable+marker+system+for+use+in+reverse+genetics+in+Plasmodium.">Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.</a> Nucleic Acids Res. 34, e39-e39 (2006).</li><li>Maier, A. G., Braks, J. A. M., Waters, A. P., Cowman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+selection+using+yeast+cytosine+deaminase/uracil+phosphoribosyl+transferase+in+Plasmodium+falciparum+for+targeted+gene+deletion+by+double+crossover+recombination.">Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination.</a> Mol. Biochem. Parasitol. 150, 118-121 (2006).</li></ol>

Die Produktion dieses Video wurde von Sigma Aldrich, Inc., die einige der Reagenzien in diesem Artikel verwendet produziert gesponsert.

Obwohl es erhebliche Fortschritte in der Entamoeba molekularbiologische Werkzeuge wurden gibt es keine aktuellen Methoden zur Verfügung, zu löschen oder zu stören Gene 4. Knockdown von Gen-Expression wurde durch die Verwendung von Haarnadel RNAs 5, small interfering RNA 6 wurde erreicht und bakteriell exprimierten dsRNA 7. Allerdings sind diese Methoden während mit Wirkung für die jeweiligen Zielgene, haben mehrere Einschränkungen, einschließlich der Verwendung von teuren Chemikalien, kontinuierliche Selektion gegen Antibiotika und die Notwendigkeit einer ständigen Überprüfung durch die hohe Inzidenz der Reversion Laufe der Zeit auftreten (Alicia Linford, persönliche Mitteilung) 8. Jede Plasmid kann in Entamoeba replizieren, da es offenbar keine strikte Reihenfolge Voraussetzung für Herkunft der DNA-Replikation 9, und so einmal transfiziert, ist es meist schwierig, ein Plasmid zu heilen. Dies schränkt die mögliche Verwendung von intakten Plasmiden in Rekombination und Gen-Knockout-Versuchen. Negative Selektionsmarker sind Schlüsselkomponenten von Gen-Targeting-Methoden, wie sie können verwendet werden, um rekombinante Subpopulationen zu beseitigen. Wir haben erfolgreich ein Codon Ausdruck optimiert FCU1 Gen in Entamoeba histolytica und getestet sein Potenzial als negative Selektionsmarker. Dieser Gen-Fusion hat für Suizid-Gen-Therapie von humanen Tumorzellen eingesetzt und metabolisiert das Prodrug 5-FC in giftige Folgeprodukte was zu einer Hemmung der RNA-und DNA-Synthese 10. FCU1 wurde vor kurzem als eine negative Selektionsmarker in Plasmodium, ein weiteres einzelligen Parasiten verwendet. Plasmodium berghei exprimieren FCU1 gefunden wurden, werden fast 1000-fach empfindlicher auf das Pro-Pharmakon in vitro und in vivo Behandlung mit 5-FC starben mehr als 99,9% der infizieren rekombinanten Parasiten in die Erythrozyten-Stufen-Maus-Modell der Malaria 11. E. histolytica exprimieren FCU1 zeigten nur eine 30-fach erhöhte Empfindlichkeit gegenüber dem Prodrug 5-FC im Gegensatz zu der Empfindlichkeit in P. beobachtet berghei. Mögliche Gründe dafür könnten großen Pool von Nukleotiden in das Nährmedium im Wettbewerb mit den toxischen Metaboliten werden. In beiden P. berghei und P. falciparum die FCU1 Marker in gezielten Gen-Unterbrechung verwendet wurde. Studien 11,12. Wir wollen die Entamoeba-Genom durch homologe Rekombination zu manipulieren. Die Validierung der FCU1/5-FC negative Selektion ist ein wichtiger Schritt in Richtung dieses Ziels.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

Entamoeba histolytica ist der Erreger der Amöbiasis und infiziert von bis zu 10% der Weltbevölkerung. Die molekulare Techniken, die die Up-und Down-Regulation der Genexpression aktiviert haben verlassen sich auf die Transfektion von Plasmiden stabil aufrechterhalten. Während diese unser Verständnis von Entamoeba Virulenzfaktoren erhöht haben, um die Kapazität exogener DNA in Genom, die es erlauben reversen Genetik Experimente, würde einen erheblichen Vorteil in der Studie von diesem Parasiten wäre zu integrieren. Die Herausforderungen durch diesen Organismus besprochen werden, Unfähigkeit, für die homologe Rekombination Ereignisse und Schwierigkeiten zu episomalen Plasmid-DNA aus transfizierten Trophozoiten Heilung zu wählen. Die späteren Ergebnisse in einem hohen Hintergrund exogener DNA, ein großes Problem bei der Identifizierung von Trophozoiten, in dem eine bona fide genomische Integration Ereignis eingetreten ist. Wir berichten über die Entwicklung einer negativen Auslese-System auf transgene Expression einer Hefe Cytosindeaminase und Uracil Phosphoribosyltransferase Chimäre (FCU1) und die Auswahl mit Prodrug 5-Fluorocytosin (5-FC) basiert. Die FCU1 Enzym wandelt ungiftig 5-FC in toxischen 5-Fluorouracil und 5-Fluoruridin-5&#39;-monophosphat. E. histolytica Linien auszudrücken FCU1 wurde festgestellt, dass 30-fach empfindlicher auf das Pro-Pharmakon im Vergleich zur Kontrollgruppe Belastung werden.

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

Wir berichten Entwicklung einer negativen Auslese-System in<em&gt; E. histolytica</em&gt; Auf transgene Expression eines chimären Proteins (FCU1) und die Auswahl mit der Prodrug 5-Fluorocytosin basiert. Die FCU1 Protein ist eine Mischung aus Hefe Cytosindeaminase und Uracil phosphoribosyltransferase. Expression von FCU1 führte zu einer erhöhten<em&gt; E. histolytica</em&gt; Sensibilität gegenüber 5-Fluorocytosin.

Entwicklung eines Negativ Selektionsmarker für Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

development-of-a-negative-selectable-marker-for-entamoeba-histolytica

Nanyang Technological University

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JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.4K Aufrufe.  University of Virginia Health System. Wir berichten Entwicklung einer negativen Auslese-System in E. histolytica Auf transgene Expression eines chimären Proteins (FCU1) und die Auswahl mit der Prodrug 5-Fluorocytosin basiert. Die FCU1 Protein ist eine Mischung aus Hefe Cytosindeaminase und Uracil phosphoribosyltransferase. Expression von FCU1 führte zu einer erhöhten E. histolytica Sensibilität gegenüber 5-Fluorocytosin.

Serie: Development of a Negative Selectable Marker for Entamoeba histolytica

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal.
Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55&deg;C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application. 
Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.

Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.

Biocontained Carcass Kompostierung für Control of Infectious Disease Outbreak in Livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

Forschung

Immunologie und Infektion

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

In-vitro-tRNA Methylierungs-Assay mit der Entamoeba histolytica DNA und tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Schnelle Identifizierung von Gram-negativen Bakterien aus Blutkulturbrühe unter Verwendung von MALDI-TOF-Massenspektrometrie

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Die Analyse der epithelialen Schäden Produziert von<em&gt; Entamoeba histolytica</em&gt; Infektion

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Der Nachweis von Human-Leukozyten-Antigen Biomarkern bei Brustkrebs Verwendung Markierungsfreie Biosensorik

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualisierung der Auswirkungen von Sputum auf Biofilm-Entwicklung eines Modells mit Kammerdeckgläser

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Verwendung von Kapseln zur Negativfärbung von Virusproben im Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...