Ein. Virus Impfung und VLPs Reinigung aus Nicotiana benthamiana Pflanzen <ol><li> Produzieren verkappt T7-RNA-Transkripte von Kartoffelvirus X (PVX)-basierten Vektorplasmide tragenden MRFV Wildtyp (wt) und Cys-mutierte Hüllprotein (CP) Gene 12, unter Verwendung Ambions T7-mMessage mMachine Kit. </li><li> Für jeden T7-Transkript Reaktion inokulieren zwei voll ausgebaut Blätter von N. benthamiana mit 10 ul Reaktionen und bebrüten die Pflanzen für 10 Tage im Gewächshaus bei 60% Luftfeuchtigkeit für 16 Stunden mit Licht (25.000-30.000 lux) bei 25 ° C und 8 h Dunkelheit bei 20 ° C. </li><li> Harves t N. benthamiana virus-infizierten Blättern 10 Tage nach der Inokulation (dpi), wiegen das Pflanzengewebe (50 Gramm) und auf Eis stellen. </li><li> Homogenisieren das Pflanzenmaterial in einem Mixer in Gegenwart von 100 ml einer gekühlten Lösung von 0,5 M Na-Citrat-Puffer, pH 5,5 (Einsatz 2 ml Puffer pro Gramm Pflanzengewebe). </li><li> Filtern Homogenat bis 3 Schichten cheeseclOTH und Klärung durch Zentrifugieren bei 27.000 × g für 30 min. Den Überstand in ein gekühltes Messzylinder, messen die Lautstärke, und dann übertragen auf eine gekühlte sterile Becher. Entsorgen Sie die Pellets. </li><li> Verfahren Überstände durch Zugabe von 10% Polyethylenglykol 8000 (PEG 8000), Rühren für 15 min bei 4 ° C und Inkubation für weitere 45 min bei 4 ° C zur Ausfällung der Virus-ähnliche Partikel (VLP)-PEG-Matrix. </li><li> Zentrifugieren der Probe bei 27.000 × g für 20 min bei 4 ° C und Entfernen des Überstandes. </li><li> Verarbeiten das Pellet durch Zugabe von 8 ml von 0,05 M Na-Citrat, pH 5,5 mit 2% Triton X-100. Schwenken Sie die Röhrchen auch zum Lösen der Pellets. Übertragen Sie die Pellet-und Puffer, um ein gekühltes sterile Becher. Spülen Sie die Röhrchen sofort mit 2 ml des gleichen Puffers und fügen Sie den Becher. Man rührt die Suspension bei 4 ° C, bis die Pellets gelöst werden (im allgemeinen 1 Std.). </li><li> Klärung durch Zentrifugation für 5 min bei 27.000 × g, verwerfen des Pellets und prozess der Überstand durch Zentrifugation bei 140.000 × g für 2 Stunden bei 4 ° C. </li><li> Resuspendieren des Pellets in 1 ml 0,05 M Na-Citrat-Puffer, pH 5,5 und platzieren der Suspension auf einem 10-40% Saccharosegradienten in 0,05 M Na-Citrat-Puffer, pH 5,5 hergestellt. </li><li> Zentrifuge die Steigung für 3 Stunden bei 140.000 xg bei 4 ° C. </li><li> Ein Licht über die Oberseite des Zentrifugenrohres und sammelt das Licht streuenden obere Bande bei 4 cm von dem oberen Ende des Rohres, verdünnt mit 0,05 M Na-Citrat-Puffer, pH 5,5, und Zentrifugieren für 3 Stunden bei 140.000 × g bei 4 ° C. </li><li> Das Pellet in 0,5 ml sterilem Wasser und bei 4 ° C für bis zu sechs Monate. </li><li> Quantifizierung der VLPs Konzentration durch Ausführen eines Bradford-Protein-Assay. Die Ausbeute an VLPs zwischen 1 und 3 g / g von Pflanzengewebe. </li></ol> 2. Fluorescein-5-Maleimid-Kennzeichnung Reaktionen von VLPs <ol><li> Eine 1 mM-Lösung von Fluorescein-5-maleimid (427,37 g / mol) in 50 mM Natrium-PhosphatE-Puffer, pH 7,0, 1 mM EDTA. Mix mit den Wirbel, und überprüfen Sie die vollständige Auflösung. Schützen Sie das Rohr aus Licht mit Aluminiumfolie. </li><li> Fügen Fluorescein-5-Maleimid zu VLPs in Schritt 1 und Mix produziert. Verwenden eines 25-fachen molaren Überschuss von Fluorescein-5-maleimid für Molmenge Sulfhydryle. Generell mit 30 ul VLPs in einer Konzentration von 1 ug / ul und 60 ul des Fluorescein-5-Maleimid Lösung erzeugt akzeptable Ergebnisse. </li><li> Inkubieren der Reaktion für 2 Stunden bei Raumtemperatur oder über Nacht bei 4 ° C. </li><li> Abbruch der Reaktion durch Zugabe von Dithiothreitol (DTT) in einer Endkonzentration von 50 mM. </li><li> Entfernen nicht umgesetzten Fluorescein unter Verwendung einer Entsalzung Spinsäule (Thermo Scientific Inc., Rockford IL) und Zentrifugation für 2 min bei 1500 xg bei Raumtemperatur. </li><li> Für SDS-PAGE-Analyse, mit 10 ul 2 x Laemmli SDS-PAGE-Probenpuffer auf 10 ul jeder Reaktion. Bewahren Sie das markierte Protein von Licht bei 4 ° C geschützt bis zu einem Monat oderin Einzel-Aliquots bei -20 ° C bis zu 3 Monaten. </li></ol> 3. Markierungsreaktion und Bestimmung von Biotin Incorporation <ol><li> Verwenden Entsalzen Spinsäulen (Thermo Scientific Inc., Rockford IL), einen Puffer Austausch von Wasser zu Phosphat-gepufferter Kochsalzlösung (PBS), pH 7,0 der VLPs in Schritt 1 erzeugten durchzuführen. </li><li> Bereiten Sie eine 20 mM Stammlösung von Maleimid-PEG2-Biotin durch Zugabe von 190 ul PBS im No-Weigh Maleimid-PEG2-Biotin und mischen Sie durch Auf-und Abpipettieren. </li><li> Fügen Maleimid-PEG2-Biotin an die VLPs und mischen. Verwenden eines 10-fachen molaren Überschuss an Reagenz zur VLP-Lösungen ≤ 2 mg / ml. </li><li> Inkubieren der Reaktion bei Raumtemperatur für 4 Stunden. </li><li> Entfernen nicht umgesetzten Maleimidbasis PEG2-Biotin unter Verwendung eines Spin-Säule entsalzt (Thermo Scientific Inc., Rockford IL) und Zentrifugation für 2 min bei 1500 xg bei Raumtemperatur. </li><li> Bestimmen Sie die Mol Biotin pro Mol VLPs mit dem Pierce Biotin Quantifizierung Kit (Thermo Scientific) und following Anweisungen des Herstellers. </li></ol> 4. Vernetzen von Cys 2-VLPs und F Peptid <ol><li> VLPs, welche im verfügbaren Cysteinreste zum Vernetzen in Schritt 2 (Cys 2-VLPs) geführt sind in der folgenden Stufe eingesetzt. Bereiten Sie eine neue Vernetzung Stammlösung (28,63 mM) durch Auflösen von 10 mg NHS-PEG4-Maleimid Vernetzer in 680 ul Dimethylsulfoxid. </li><li> Man löst eine 17 (F)-Aminosäuren langes Peptid (LLPNMOKDKEACAKAPL) in 50 ul Konjugationspuffer (1 x PBS, pH 7,2, 1 mM EDTA) in einer Konzentration von 0,1 mM. </li><li> Add 4 ul Quervernetzer bis 50 ul des gelösten Peptid (Protein-NH2) bei 1 mM Endkonzentration (das ist ein 10-facher molarer Überschuß für 0,1 mM Peptidlösung). </li><li> Inkubieren der Reaktion für 2 h bei 4 ° C. </li><li> Läutern vernetzten Peptid unter Verwendung einer Entsalzungssäule (Thermo Scientific Inc., Rockford IL), äquilibriert mit Konjugationspuffer (1 x PBS, pH 7,2, 1 mM EDTA) durch Zentrifugation bei 1.500 xg1 min bei Raumtemperatur. </li><li> Einrichten einer Reaktionsmischung kombiniert VLPs (Protein-SH) und vernetztes Peptid (Protein-NH2) in einem molaren Verhältnis der Anzahl von Thiolen und Aminen aktiviertes in der Reaktion und die Differenz im Molekulargewicht zwischen VLPs und Peptid beteiligt bestimmt. </li><li> Inkubieren der Reaktionsmischung bei Raumtemperatur für 2 h bei 4 ° C. </li><li> Für die Western-Blot-Analyse, mischen Sie die resultierende konjugierte Produkt 1:1 mit Laemmli-Puffer, kochen für 10 min, und fahren Sie mit SDS-PAGE auf einem pre-cast 10-20% Tris-Glycin-Gel (Invitrogen), gefolgt von Elektroblotten auf eine Nitrocellulosemembran (Invitrogen). Blockieren der Membranen mit 1 x PBS, pH 7,2, enthaltend 0,05% Tween-20 (PBS-Tween) und 5% Magermilchpulver und Sonde mit Peptid-spezifischen Antikörper, die in geeigneten Phosphatase-markierten sekundären Antikörper folgt. </li></ol>

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Das hier vorgestellte Verfahren erlaubt eine sehr empfindliche und schnelle Analyse von reaktiven Cysteine ​​auf der Oberfläche der Pflanzen produzierte VLPs sowie auf anderen Proteinkomplexen. Maleimide sind Thiol-spezifische Reagenzien, die mit freien Sulfhydryl enthaltenden Molekülen reagieren, um eine stabile Thioether-Bindungen zu bilden. Diese Methode stützt sich auf die Spezifität der Maleimide mit Sulfhydrylgruppen nicht in Wechselwirkungen mit anderen Aminosäuren beteiligt reagieren. Die Erhaltung der ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalognummer </td><td> Kommentare </td></tr><tr><td> Thinwall, Ultra-Clear Tubes </td><td> Beckman </td><td> 344059 </td><td></td></tr><tr><td> mMessage mMachine T7 Kit </td><td> Leben Tecnologies </td><td> AM1344M </td><td></td></tr><tr><td> Fluorescein-5-maleimid </td><td> Thermo Scientific Life Technologies </td><td> 46130 F150 </td><td> 46130 ist nicht in Ordnung Ersatz für F150 </td></tr><tr><td> Pierce Biotin Quantifizierungskit </td><td> Thermo Scientific </td><td> 28005 </td><td></td></tr><tr><td> EZ-Link Maleinimid-PEG2-Biotin, No-Wiegen Format </td><td> Thermo Scientific </td><td> 21901 </td><td></td></tr><tr><td> SM (PEG) n Vernetzer </td><td> Thermo Scientific </td><td> 22107 </td><td></td></tr><tr><td> 10-20% Tris-Glycin-Gel </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Laemmli Buffer </td><td> Bio-Rad </td><td> 1610737 </td><td></td></tr><tr><td> Tris Glycine SDS Laufpuffer </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> Tris Glycine Transfer-Buffer </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> Nitrocellulose Membrane Filter Paper Sandwich </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> Phosphatase markiert Affinity Purified Antibody to Kaninchen IgG </td><td> Kirkegaard und Perry Laboratories </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP Phosphatasesubstrat </td><td> Kirkegaard und Perry Laboratories </td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

Imitieren und Nutzung Virus Eigenschaften und physikalisch-chemischen und physikalischen Eigenschaften verspricht, um Lösungen für einige der weltweit dringlichsten Herausforderungen. Die schiere Reichweite und Arten von Viren mit ihren faszinierenden Eigenschaften gekoppelt potenziell geben unendlich viele Möglichkeiten für Anwendungen in der Virus-basierten Technologien. Viren haben die Fähigkeit, zu Teilchen mit diskreten Form und Größe, Spezifität Symmetriegründen polyvalence und stabile Eigenschaften in einem weiten Bereich von Temperatur und pH-Bedingungen selbst zusammen. Es überrascht nicht, mit einer solchen Reihe von bemerkenswerten Eigenschaften, werden Viren zur Verwendung in Biomaterialien 9, Impfstoffen 14, 15, elektronische Materialien, chemische Werkzeuge und molekularen elektronischen Behälter 4, 5, 10, 11, 16, 18, ​​12 vorgeschlagen. Um Viren in der Nanotechnologie zu verwenden, müssen sie aus ihren natürlichen Formen modifiziert werden, um neue Funktionen zu verleihen. Diese anspruchsvolle prozess kann durch verschiedene Mechanismen, einschließlich genetischer Modifikation des viralen Genoms und chemisch Anbringen fremden oder gewünschten Moleküle an die Viruspartikel reaktiven Gruppen 8 durchgeführt werden. Die Fähigkeit, einen Virus zu modifizieren vorwiegend abhängig von den physikalisch-chemischen und physikalischen Eigenschaften des Virus. Darüber hinaus müssen die genetische oder physiochemischen Modifikationen ohne Beeinträchtigung der nativen Struktur Virus und Virus-Funktion ausgeführt werden. Maize rayado fino Virus (MRFV) Hüllproteinen in Escherichia coli Herstellung stabiler und leere VLPs, die durch Protein-Protein stabilisiert sind selbstorganisieren Interaktionen und die in Virus-basierten Technologien Anwendungen 8 verwendet werden. VLPs in Tabakpflanzen hergestellt wurden als ein Gerüst, auf dem eine Vielzahl von Peptiden kovalent angezeigt werden 13 untersucht. Hier beschreiben wir die Schritte 1) bestimmen, welche der Lösungsmittel-zugänglichen Cysteine ​​in einem Virus-Kapsid sind für ModifikationenKation, und 2) Bioconjugate Peptide an den modifizierten Kapside. Durch die Verwendung von nativem oder durch Mutation eingefügten Aminosäurereste und Standardkoppelstellen Technologien haben eine breite Vielfalt von Materialien auf der Oberfläche von Pflanzenviren wie Brome Mosaic Virus 3, Carnation-Mottle-Virus 12, Cowpea chlorotic mottle virus 6, Tabak Mosaik angezeigt worden Virus 17, Turnip yellow mosaic virus 1 und MRFV 13.

Transiente Expression von mutierten MRFV Hüllproteins (CP) Gene in N. benthamiana Pflanzen in einem PVX-basierten Vektor Herstellung VLPs ist in Abbildung 1 beschrieben. Das modifizierte MRFV Hüllproteingen wird durch PCR amplifiziert und dann platziert unter der transkriptionellen Kontrolle des duplizierten subgenomische CP-Promotor in einem PVX-basierten Vektor, pP2C2S 2, (ein Geschenk von D. Baulcombe, Sainsbury Laboratories, Norwich, England). Im vitro RNA-Transkriptio...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Ein Verfahren, um das Lösungsmittel Zugänglichkeit der Thiolgruppe von Cystein-Resten der Analyse<em&gt; Mais rayado fino-Virus</em&gt; (MRFV)-Virus-ähnliche Partikel (VLP) durch ein Peptid Vernetzungsreaktion folgt beschrieben. Das Verfahren nutzt die Verfügbarkeit von mehreren chemischen Gruppen auf der Oberfläche der VLPs, die als Ziele für spezielle Reaktionen sein kann.

Analyse der Solvent Zugänglichkeit des Cystein Rückstände auf<em&gt; Mais rayado fino-Virus</em&gt; Virus-ähnliche Partikel Produziert in<em&gt; Nicotiana benthamiana</em&gt; Pflanzen und Vernetzung der Peptide an VLPs

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K Aufrufe.  Agricultural Research Service, United States Department of Agriculture. Ein Verfahren, um das Lösungsmittel Zugänglichkeit der Thiolgruppe von Cystein-Resten der Analyse Mais rayado fino-Virus (MRFV)-Virus-ähnliche Partikel (VLP) durch ein Peptid Vernetzungsreaktion folgt beschrieben. Das Verfahren nutzt die Verfügbarkeit von mehreren chemischen Gruppen auf der Oberfläche der VLPs, die als Ziele für spezielle Reaktionen sein kann.

Serie: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Isolation und Genomanalyse Einzel Virionen mit &#39;Single Virus Genomics&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Forschung

Immunologie und Infektion

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 ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In-vitro-Analyse Myd88-vermittelte zelluläre Immunantwort gegen das West-Nil-Virus-Infektion Mutantenstamm

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modellierung des Lebenszyklus von Ebola Virus unter Biosafety Level 2 Bedingungen mit Virus-ähnlichen Partikeln, die Tetracistronic Minigenome

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

Die Nutzung der Soft-Agar-Overlay-Technik zur Leinwand für bakteriell Hemmstoffe Produziert

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

Kombination von Analyse von DNA in eine grobe Virion-Extraktion mit der Analyse der RNA aus den infizierten Blättern, entdecken neue Virus-Genom

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Gründung der Virusinfektion und Analyse von Host-Virus-Wechselwirkung in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

Durchflusszytometrischen Durchflussmessung von ROS-Produktion In Makrophagen als Reaktion auf FcγR Vernetzung

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...