Minimal data set

Minimal data set for 2017 PLOS ONE article "Serum copeptin and neuron specific enolase are markers of neonatal distress and long-term neurodevelopmental outcome"

Clinical characteristics of the study groups.

<p>Clinical characteristics of the study groups.</p

Copeptin and NSE concentrations in serum samples over time (n = 75).

<p>(A, B) Copeptin levels decrease over time, with highest levels measured at 6 hours after birth. Copeptin concentrations are shown for individual neonates (A) as well as the median ±IQR concentration for each time point (B). Compared to the 6 hour time point, concentrations were significantly lower at all subsequent time points (p = 0.0448 at 6 vs. 12 hours, and p < 0.0001 at all other time points). (C, D) NSE levels are relatively stable until 24 h after birth, after which the levels decrease. Data is shown for individual neonates (C) and as the median ± IQR at each time point (D). Compared to the 6 hour time point, concentrations were significantly lower only at 168 hours after birth (p = 0.0013). The Kruskal-Wallis test with Dunn’s multiple comparisons test was used.</p

Copeptin and NSE levels after birth in relation to two-year outcome.

<p>(A) Copeptin levels were significantly higher in neonates in the poor outcome group (n = 25) than in the favorable outcome group (n = 50) at 6 hours (p = 0.0068), 12 hours (p = 0.0050) and 48 hours (p = 0.0226) after birth. (B) NSE levels were significantly higher in the neonates in the poor outcome group compared to the favorable outcome group at all time points (p = 0.0009 at 6 hours, p = 0.0005 at 12 hours, p = 0.0005 at 24 hours, p = 0.0009 at 48 hours, and p = 0.0029 at 72 hours). (C-D) ROC curves of combined copeptin and NSE concentrations at 6 hours (C) and 12 hours (D) after birth in relation to two year neurodevelopmental outcome. Graphs A and B show the median ± IQR. The data for each time point were analyzed separately with the Mann-Whitney U-test, and the Holm-Sidak method was used to compute thresholds to ensure a family-wise error rate below 0.05.</p

Copeptin levels in relation to therapeutic hypothermia.

<p>Copeptin concentrations after birth were lower in the hypothermic neonates from the TOBY cohort (n = 11) compared to the normothermic neonates from the same cohort (n = 10) at 6 hours after birth (p = 0.0495). Graph shows the median ± IQR. The data for each time point was analyzed separately with the Mann-Whitney U-test, and the Holm-Sidak method was used to compute thresholds to ensure a family-wise error rate below 0.05.</p

Kinetics of PKCε activation by VHHs A10, C1 and D1.

<p>The kinase activity of full-length PKCε in the presence (A) and absence (B) of PKC activators DOG and PS was measured with varying MARCKS substrate concentrations. The VHH concentration was constant (1 µg/well) for each experiment. The data is presented as percentage maximal control activity (control activity with 1000 µM substrate) ± SEM and represents at least 3 independent experiments, each with duplicates. Note that the V_max values for the VHHs have not been reached yet, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035630#pone-0035630-t002" target="_blank">table 2</a> for analysis.</p

SPR sensograms and fits for PKCε activating VHHs.

<p>SPR sensograms and fits for second-order Langmuir binding models are shown for VHHs A10 (A), C1 (B) and D1 (C). The VHH injection time was 3 min, followed by a dissociation time of 5 min. The surface was regenerated with an injection of 10 mM NaOH for 3 min, followed by a stabilization time of 5 min between each VHH injection. Five concentrations of each VHH were used, with the middle concentration injected twice as an internal control. The VHH concentrations (in µg/ml) are marked adjacent to each fit on the right hand side of the figure.</p

Kinetics of PKCε inhibition by VHHs E6 and G8.

<p>(A–B) The kinase activity of full-length PKCε in the presence (A) and absence (B) of PKC activators DOG and PS was measured with varying MARCKS substrate concentrations. (C) The kinase activity of the catalytic domain of PKCε was measured with varying MARCKS substrate concentrations. The VHH concentration was constant (1 µg/well) for each experiment. The data is presented as percentage maximal control activity (control activity with 1000 µM substrate) ± SEM and represents at least 3 independent experiments, each with duplicates. The catalytic domain activity (C) with G8 is an exception with only 2 independent experiments with duplicates.</p

Association and dissociation constants for the interaction of VHHs with PKCε obtained from SPR measurements.

<p>Association and dissociation constants for the interaction of VHHs with PKCε obtained from SPR measurements.</p

PKCε in rat brain extract.

<p>(A) 15 µg of rat brain extract was separated on a SDS-PAGE gel. PKCε was detected with anti-PKCε and HRP-conjugated goat anti-mouse antibodies. (B) Immunoprecipitations were performed with rat brain extract using a commercial anti-PKCε antibody (IgG Ab) and VHHs. PKCε (marked with an arrowhead) is visible at 90 kDa on lane 1. The bands at 55 kDa and 25 kDa on lane 1 represent the heavy and light chains of the anti-PKCε antibody. The bands at 16 kDa for A10, C1, D1, E6 and G8 represent the VHHs. A sample of uncoated protein A sepharose beads was included as a negative control (lane 2 = ctrl).</p