The influence of acetate, MOPS and osmolytes on the levels of non-dividing and persister cells.

<p>Bacteria were grown aerobically for 24 h at 37°C in LB medium supplemented with 0.2% acetate, 40 mM MOPS pH 7.4, 0.2% glucose, 0.2% glycerol, 0.2% trehalose or 0.2% betaine. The control culture was grown in LB without supplements. (A) The total number of cells per ml was obtained using a hemocytometer. Dividing cell counts were estimated by plating serial dilutions on LA. The number of non-dividing cells was calculated by subtracting colony-forming units counts from the total number of cells. (B) <i>E. coli</i> stationary- phase cells grown in the presence of glucose are larger than control cells. 1000×magnification. (C) To isolate and estimate persisters the stationary cultures were diluted to an OD₅₉₅ = 0.1 and exposed to ampicillin (200 µg/ml) for 10 h at 37°C. Antibiotic- tolerant bacteria were plated for colony counting. 100% corresponds to the total number of cells (dividing and non-dividing) before antibiotic treatment. Means and standard deviation of three independent experiments are shown.</p

The level of ATP and membrane stability in <i>E. coli</i> stationary-phase cultures.

<p>The cultures were grown as described in the legend to Figue 4. (A) ATP level was determined using BacTiter Glo chemicals (Promega). (B) Membrane stability was assessed after staining cells with the LIVE/DEAD assay solution (Invitrogen). Error bars represent standard deviation of three independent experiments. RFU, relative fluorescence units.</p

Stationary-phase <i>E. coli</i> cultures that accumulate protein aggregates produce increased levels of persisters.

<p>(A) Growth curves of <i>E. coli</i> MC4100. The bacteria were grown aerobically (circles) or anaerobically (triangles) at 37°C in LB medium supplemented (black symbols) or not (white symbols) with 0.2% glucose. (B) Protein aggregates were isolated from the stationary-phase cultures as described in Materials and Methods. Equal volumes of insoluble fractions isolated in the sucrose gradient were resolved by SDS-PAGE and visualized by Coomassie staining. (C) After 24 h the cultures were diluted to an OD₅₉₅ = 0.1 and exposed to ampicillin (200 µg/ml) for 10 h at 37°C. Antibiotic- tolerant bacteria were plated for colony counting at the times indicated in the graph. The error bars indicate the standard deviations of three independent experiments.</p

Aggregation of proteins, formation of persisters and oxidation of proteins in the presence of acetate, MOPS and osmolytes.

<p>Bacteria were grown aerobically for 24 h at 37°C in LB medium supplemented with 0.2% acetate, 40 mM MOPS pH 7.4, 0.2% glucose, 0.2% glycerol, 0.2% trehalose or 0.2% betaine, unless otherwise stated. The control culture was grown in LB without supplements. Protein aggregates were isolated from 24 h cultures, resolved by SDS-PAGE and visualized by Coomassie staining. Oxidized, DNPH-derivatized proteins were resolved by SDS-PAGE and immunodetected using anti-dinitrophenol antibodies (E). The amount of aggregated proteins in relation to total protein in cell extracts (A) and the relative level of oxidized proteins (C) were calculated on the basis of densitometry. (B) After 24 h the cultures were diluted to an OD₅₉₅ = 0.1 and exposed to ampicillin (200 µg/ml) for 10 h at 37°C. Antibiotic- tolerant bacteria were plated for colony counting at the times indicated in the graph. 100% corresponds to the number of colonies before antibiotic treatment. Means and standard deviation of three independent experiments are shown. (D) Representative growth curves of selected cultures are shown. (E) Samples corresponding to the same OD₅₉₅ were loaded on the gels. Lane 1- the control culture, lane 2- LB+acetate, lane 3 - LB +MOPS, lane 4 - LB+trehalose, lane 5- LB+glucose. The asterics indicates EF-Tu, the most abundant protein in the aggregates.</p

Baseline characteristics of study participants.

<p>Baseline characteristics of study participants.</p

Bioavailability of ticagrelor and AR-C124910XX over time in patients with STEMI and NSTEMI.

<p>Plasma concentrations of (A) ticagrelor and (B) AR-C124910XX during the first 6 h after oral administration of a 180 mg ticagrelor loading dose in patients with STEMI and NSTEMI. NSTEMI: non-ST-elevation myocardial infarction, STEMI: ST-elevation myocardial infarction.</p

Prevalence of high platelet reactivity over time in STEMI and NSTEMI patients.

<p>Proportion of patients with high platelet reactivity assessed with (A) the VASP assay and Multiplate (B) at baseline, and at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, and 12 h after administration of a 180 mg ticagrelor loading dose in patients with STEMI and NSTEMI. HPR: high platelet reactivity, NSTEMI: non-ST-elevation myocardial infarction, STEMI: ST-elevation myocardial infarction, VASP: vasodilator-stimulated phosphoprotein.</p

Platelet reactivity over time in STEMI and NSTEMI patients.

<p>Platelet reactivity evaluated with (A) the VASP assay and (B) Multiplate at baseline, and at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, and 12 h after administration of a 180 mg ticagrelor loading dose in patients with STEMI and NSTEMI. NSTEMI: non-ST-elevation myocardial infarction, STEMI: ST-elevation myocardial infarction, VASP: vasodilator-stimulated phosphoprotein.</p