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Peter Veranič - One of the best experts on this subject based on the ideXlab platform.

  • Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-McHerry protein.
    PloS one, 2014
    Co-Authors: Matej Skočaj, Nataša Resnik, Maja Grundner, Katja Ota, Nejc Rojko, Vesna Hodnik, Gregor Anderluh, Andrzej Sobota, Peter Maček, Peter Veranič
    Abstract:

    Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with McHerry (OlyA-McHerry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-McHerry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-McHerry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-McHerry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-McHerry in comparison with these other proteins. Furthermore, we show that OlyA-McHerry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-McHerry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-McHerry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

  • tracking cholesterol sphingomyelin rich membrane domains with the ostreolysin a McHerry protein
    PLOS ONE, 2014
    Co-Authors: Matej Skočaj, Nataša Resnik, Maja Grundner, Katja Ota, Nejc Rojko, Vesna Hodnik, Gregor Anderluh, Andrzej Sobota, Peter Maček, Peter Veranič
    Abstract:

    Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with McHerry (OlyA-McHerry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-McHerry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-McHerry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-McHerry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-McHerry in comparison with these other proteins. Furthermore, we show that OlyA-McHerry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-McHerry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-McHerry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Matej Skočaj - One of the best experts on this subject based on the ideXlab platform.

  • Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-McHerry protein.
    PloS one, 2014
    Co-Authors: Matej Skočaj, Nataša Resnik, Maja Grundner, Katja Ota, Nejc Rojko, Vesna Hodnik, Gregor Anderluh, Andrzej Sobota, Peter Maček, Peter Veranič
    Abstract:

    Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with McHerry (OlyA-McHerry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-McHerry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-McHerry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-McHerry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-McHerry in comparison with these other proteins. Furthermore, we show that OlyA-McHerry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-McHerry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-McHerry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

  • tracking cholesterol sphingomyelin rich membrane domains with the ostreolysin a McHerry protein
    PLOS ONE, 2014
    Co-Authors: Matej Skočaj, Nataša Resnik, Maja Grundner, Katja Ota, Nejc Rojko, Vesna Hodnik, Gregor Anderluh, Andrzej Sobota, Peter Maček, Peter Veranič
    Abstract:

    Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with McHerry (OlyA-McHerry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-McHerry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-McHerry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-McHerry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-McHerry in comparison with these other proteins. Furthermore, we show that OlyA-McHerry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-McHerry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-McHerry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Martin Palus - One of the best experts on this subject based on the ideXlab platform.

  • development and characterization of recombinant tick borne encephalitis virus expressing McHerry reporter protein a new tool for high throughput screening of antiviral compounds and neutralizing antibody assays
    Antiviral Research, 2021
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that McHerry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the McHerry-based neutralization test. Collectively, recombinant McHerry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

  • Development and characterization of recombinant tick-borne encephalitis virus expressing McHerry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.
    Antiviral research, 2020
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus, Xavier De Lamballerie
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over

Kentaro Yoshii - One of the best experts on this subject based on the ideXlab platform.

  • development and characterization of recombinant tick borne encephalitis virus expressing McHerry reporter protein a new tool for high throughput screening of antiviral compounds and neutralizing antibody assays
    Antiviral Research, 2021
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that McHerry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the McHerry-based neutralization test. Collectively, recombinant McHerry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

  • Development and characterization of recombinant tick-borne encephalitis virus expressing McHerry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.
    Antiviral research, 2020
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus, Xavier De Lamballerie
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over

Jan Haviernik - One of the best experts on this subject based on the ideXlab platform.

  • development and characterization of recombinant tick borne encephalitis virus expressing McHerry reporter protein a new tool for high throughput screening of antiviral compounds and neutralizing antibody assays
    Antiviral Research, 2021
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that McHerry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the McHerry-based neutralization test. Collectively, recombinant McHerry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

  • Development and characterization of recombinant tick-borne encephalitis virus expressing McHerry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.
    Antiviral research, 2020
    Co-Authors: Jan Haviernik, Ludek Eyer, Kentaro Yoshii, Shintaro Kobayashi, Jiri Cerny, Antoine Nougairede, Jeanselim Driouich, Jiri Volf, Martin Palus, Xavier De Lamballerie
    Abstract:

    The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the McHerry reporter protein. The resulting TBEV reporter virus (named McHerry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the McHerry-TBEV virus produced smaller plaques. The magnitude of McHerry expression correlated well with progeny virus production but remained stable over