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Accumulation Assay

The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform

C K Stover – 1st expert on this subject based on the ideXlab platform

  • development of a liquid chromatography mass spectrometry based drug Accumulation Assay in pseudomonas aeruginosa
    Analytical Biochemistry, 2009
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [3H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

  • Development of a liquid chromatography/mass spectrometry-based drug Accumulation Assay in Pseudomonas aeruginosa.
    Analytical Biochemistry, 2008
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonas aeruginosa , rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC 50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3 H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [ 3 H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

Kelly A Rose – 2nd expert on this subject based on the ideXlab platform

  • development of a liquid chromatography mass spectrometry based drug Accumulation Assay in pseudomonas aeruginosa
    Analytical Biochemistry, 2009
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [3H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

  • Development of a liquid chromatography/mass spectrometry-based drug Accumulation Assay in Pseudomonas aeruginosa.
    Analytical Biochemistry, 2008
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonas aeruginosa , rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC 50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3 H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [ 3 H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

Steve Dunham – 3rd expert on this subject based on the ideXlab platform

  • development of a liquid chromatography mass spectrometry based drug Accumulation Assay in pseudomonas aeruginosa
    Analytical Biochemistry, 2009
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [3H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.

  • Development of a liquid chromatography/mass spectrometry-based drug Accumulation Assay in Pseudomonas aeruginosa.
    Analytical Biochemistry, 2008
    Co-Authors: Kelly A Rose, Lanhsin Liang, Steve Dunham, C K Stover

    Abstract:

    Abstract Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonas aeruginosa , rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC 50 values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/Accumulation Assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence Assay was established to measure the time-dependent Accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM–MexCDOprJ–MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP Accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established Assay conditions were applied to a radiolabeled Assay format using 3 H-labeled ciprofloxacin. At the concentration tested, the Accumulation of [ 3 H]ciprofloxacin approached a plateau after 15 min and the amount of Accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based Assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.