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  • Desflurane concentrations in arterial blood

    2018-11-14

    Desflurane concentrations in arterial blood samples were analyzed by Micropore Membrane Inlet Mass Spectrometry (MMIMS). Data on arterial blood desflurane during uptake and elimination are displayed in Fig. 3. The infusion of MCh induced bronchoconstriction and impaired arterial oxygenation did not affect desflurane pharmacokinetics.
    Experimental design, materials and methods
    Acknowledgments
    Value of the data
    Data The raw data files (fastq files) that were used in the analysis and interpretation in [2–4] are available in European Bioinformatics Institute. Accession number E-MTAB-2017. http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2017/. Sample IDs are:
    Materials and methods Corynebacterium pseudotuberculosis strain 1002 [1] grown in petri dishes containing BHI media (broth composed of (g/L): calf-brain infusion 200.00, beef-heart infusion 250.00, proteose peptone 10.00, dextrose 2.00, sodium chloride 5.00, di-sodium phosphate 2.50pH 7.4±0.2 at 25°C) at room temperature (RT). One colony was used to prepare the pre-inoculum in 20mL of BHI media supplemented with 0.05% Tween 80. The culture was grown overnight at 37°C in a shaker at 160rpm. One millilitre of this pre-inoculum was used to prepare the inoculum in an Erlenmeyer flask containing 100mL fresh BHI, and this culture was incubated at 37°C at 160rpm. This process was monitored until the beginning of the exponential growth phase (A600=0.2), which was reached approximately 2.5h after the initial inoculation [3]. After the culture reached the beginning of the exponential growth phase, the inoculum was divided into 4 50-mL Falcon tubes (1 for each condition), each containing a final volume of 20mL, and these tubes were then centrifuged for 3min at 8000rpm at RT. The pellet was resuspended in fresh BHI specific to each condition. For the AUY922 stress condition, the media was supplemented with hydrochloric acid (in which the pH changed to 5). Osmotic stress was achieved with 2M NaCl, and thermal stress was induced by resuspending the pellet in BHI medium pre-heated to 50°C. In the control condition, bacterial pellets were resuspended in BHI medium at a physiological condition. After the addition of culture media, the tubes were kept in a shaker at 37°C and 160rpm for 15min, with the exception of the thermal stress sample that was subjected to a temperature of 50°C. An aliquot of each condition was used for decimal dilutions from 10−1 to 10−6, from which 10−4 to 10−6 bacteria were seeded in BHI agar, and petri dishes were kept at 37°C for 48h for viability analysis and colony counting (this step was performed in duplicate). The remaining sample was subjected to centrifugation at RT for 3min at 8000rpm, and the pellet was resuspended in 2ml of RNAlater, according to the manufacturer’s instructions [3]. The bacteria suspended in RNAlater® buffer were subjected to total RNA extraction using the ChargeSwitch® total RNA cell kit (Invitrogen, USA) in accordance with the manufacturer’s recommendations, including the following adaptations: after the addition of the lysis buffer (Invitrogen), the material was transferred to 2-mL tubes partially filled with 1-mm diameter glass microbeads (Bertin Technologies). The cells were lysed mechanically using a Prescellys 24 homogeniser, set at 6500rpm, for 2 cycles (15s per cycle) with an interval of 30s between the cycles. The samples were centrifuged for 1min, and the supernatant was transferred to fresh 2-ml tubes and incubated in a dry bath at 60°C for 15min (represents the complete original protocol). DNase was added to eliminate the residual genomic DNA. The elution of the total RNA from the magnetic beads was performed using 100μL of milli-Q RNase-free water. The amount of total RNA was obtained by Qubit® 2.0 fluorometer (Invitrogen) [3]. To enrich the mRNA, rRNA from each total RNA sample was removed by Ribominus™ Transcriptome Isolation kit for yeast and bacteria (Invitrogen, USA), in accordance with the manufacturer’s recommendations. The rRNA-depleted RNA was used for cDNA synthesis using the SOLiD™ Total RNA-Seq kit in accordance with the standard protocol recommended by the manufacturer, and quantification was performed by Qubit® 2.0 fluorometer (Invitrogen) [3].