Multiple model simulations with varied parameter sets represented the experimental cell-to-cell heterogeneity.

<p>(A-E) Cell-to-cell heterogeneity was modelled by varying parameter values and input characteristics (magnitude and duration of the applied calcium influx) and performing multiple simulations. (A) The median and inter-quartile regions of all model inputs (solid black line within dark grey region) well resembled the median and inter-quartile regions (black dotted line within light grey region) of previously published fluorescent measurements of cytosolic calcium [Ca_c; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]]. (B-E) For each simulation, the metrics shown in (B-E) were calculated. The coloured data points link these simulations across the figures. The green data points were predicted with the parameter set as listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">2</a>. The parameter sets predicting the yellow, cyan and red data points are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s003" target="_blank">S2 Table</a>. (B,C) The predicted variability in the (B) maximum fold change and (C) recovery duration of the model input (black box) closely matched experimental measurements (white box). Recovery duration was calculated as the time taken for the signal to recover to ±2% of baseline signal. (D) Box- and scatter-plots of the minimum ATP, maximum AMPK activity and maximum glucose fold changes during the excitotoxic stimulus, calculated from multiple model predictions and experimental measurements (Exp.) from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]. (E) Box- and scatter-plots of the post-excitotoxicity recovery duration of the ATP, AMPK activity and glucose levels as calculated from model predictions and experimental measurements (Exp.) from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>].</p

Parameter constraints determined from steady-state (ss) analysis.

<p>Parameter constraints determined from steady-state (ss) analysis.</p

The computational model correctly predicted ATP, AMPK activity and glucose dynamics measured in neurons exposed to glutamate.

<p>(A) Model schematic. State variables are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">Table 1</a>, and reaction numbers correspond to those listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s002" target="_blank">S1 Table</a>. (B-E) Model input and simulations (solid black lines) overlaid on the median and inter-quartile regions (dotted black line, grey shaded area) of previously published fluorescence measurements in single cerebellar granule neurons exposed to glutamate for 10 min [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]. The time of stimulus (model input or glutamate exposure) is marked with a light grey bar. Values were normalised to baseline. (B) A transient (10 min) increase in cytosolic calcium was applied as model input (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#sec008" target="_blank">Methods</a> and d[Cac]/dt equation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s002" target="_blank">S1 Table</a>), and fitted to fluorescence measurements of cytosolic calcium (Fluo-4 AM) in CGNs exposed to glutamate. (C) The simulated ATP dynamics closely aligned with experimental measurements of intracellular ATP concentration [ATeam is a fluorescent reporter of intracellular ATP concentration; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref016" target="_blank">16</a>]]. (D) The simulated transient activation of AMPK resembled experimental measurements of AMPK activity [AMPKAR is a fluorescent reporter of AMPK activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref017" target="_blank">17</a>]]. (E) The model also correctly predicted a prolonged elevation of intracellular glucose and its delayed recovery [Glucose-FRET is a fluorescent reporter of intracellular glucose concentration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref018" target="_blank">18</a>]].</p

The computational model can represent the bioenergetic collapse induced by severe excitotoxicity, modelling neuronal necrosis.

<p>We investigated the predicted responses following more severe excitotoxic stimuli by increasing the (A-C) duration or (D-F) magnitude of the applied calcium influx (model input). (A) Prolonging the Ca_c influx beyond 5 min did not predict further depletion of ATP beyond a minimal level (grey dots). In contrast, longer periods of Ca_c influx were predicted to speed up ATP recovery (black dots). The calcium influx for which the graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.g001" target="_blank">Fig 1</a> were plotted is indicated with a black dashed line. (B) ATP was predicted to initiate recovery during longer periods of Ca_c influx. (C) The predicted ATP dynamics (black line) during a 60 min excitotoxic stimulus were validated with <i>de novo</i> fluorescent measurements of ATP concentration (ATeam) in CGNs exposed to glutamate for 60 min (grey lines) (D) More severe calcium influx was predicted to exacerbate ATP depletion (grey dots) and prolong its recovery (black dots). Behaviour consistent with neuronal necrosis was predicted for maximal values of calcium influx > 7 fold change, where the ATP concentration collapsed to ~0, and did not recover. (E) The model predicted characteristic switch-like behaviour in the ATP dynamics leading to necrotic energetic collapse on severe calcium influx. (F) At magnitudes of calcium influx that induced ATP collapse (lighter grey lines), the model also predicted altered calcium dynamics. (G) The steady-state concentrations of ATP and Ca_c well predicted the simulation outcome (viable/necrosis) following calcium influx. (H) The percentage of neurons predicted to undergo necrosis or to remain viable following an excitotoxic stimulus were similar to levels measured in populations of cortical neurons exposed to NMDA. Experimental data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref037" target="_blank">37</a>] Fig 1F, and the bar charts display mean ± SEM.</p

Reaction (Rx) equations and kinetic flux values for the modelled reaction network.

<p>Reaction (Rx) equations and kinetic flux values for the modelled reaction network.</p

The BAX/BAK-like protein BOK is a prognostic marker in colorectal cancer

Abstract The intrinsic or mitochondrial apoptosis pathway is controlled by the interaction of antiapoptotic and pro-apoptotic members of the BCL-2 protein family. Activation of this death pathway plays a crucial role in cancer progression and chemotherapy responses. The BCL-2-related ovarian killer (BOK) possesses three BCL-2 homology domains and has been proposed to act in a similar pro-apoptotic pathway as the pro-apoptotic proteins BAX and BAK. In this study, we showed that stage II and III colorectal cancer patients possessed decreased levels of BOK protein in their tumours compared to matched normal tissue. BOK protein levels in tumours were also prognostic of clinical outcome but increased BOK protein levels surprisingly associated with earlier disease recurrence and reduced overall survival. We found no significant association of BOK protein tumour levels with ER stress markers GRP78 or GRP94 or with cleaved caspase-3. In contrast, BOK protein levels correlated with Calreticulin. These data indicate BOK as a prognostic marker in colorectal cancer and suggest that different activities of BOK may contribute to cancer progression and prognosis

Steady-state concentrations of modelled state variables.

<p>Steady-state concentrations of modelled state variables.</p

Sensitivity analysis indicated that glucose import dynamics are critical to the post-excitotoxic glucose recovery.

<p>(A-C) Parameters were varied by 0.5 (navy), 0.75 (light blue), 1 (green), 1.5 (orange) and 2 (red) times the steady-state values listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">2</a>, and the effect was calculated for the post-excitotoxic recovery duration of the (A) ATP, (B) AMPK activity and (C) Glucose signals. The varied parameter is written under each bar chart. The 5 parameters with the greatest effect on each metric are shown (left to right in order of effect), along with other parameters mentioned in the text. Data were omitted for parameter values at which the modelled state variables did not return to baseline within the simulation time (100 min). (D, E) Experimental traces (top panels) and multiple model simulations (bottom panels) of intracellular glucose concentration with either (D) glucose import or (E) AMPK inhibited prior to exposure to a transient excitotoxic stimulus. (D) Glucose import was inhibited by exposure to Cytochalasin B or by reduction of the modelled glucose import kinetics (Rx 9). (E) AMPK activity was inhibited by exposure to Compound C or by reduction of the modelled AMPK phosphorylation kinetics (Rx 6). Compound C experiments have been published previously [Fig 6A from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]]. (F) Box- and scatter-plots of the glucose recovery duration with and without glucose import or AMPK inhibition (* ranksum p < 0.05).</p

Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy

This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsiveto an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy