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The time course of the similarities for the sets of dispensable reactions. In addition, for both conditions, the peak in dissimilarity between sets of EFMs occurs between 30 and 50 minutes. Between 50 and 90 minutes, both condition-specific networks exhibit similarity with respect to their sets of EFMs and sets of dispensable reactions, demonstrating that the system has started settling in the new condition.Fractional appearance of reactions in EFMs discriminates two types of reactionsbetween reactions. A heatmap representation of the correlation matrix is shown in the Additional file 1: Figure S1. The results hint at the presence of different temporal patterns apparent from the clustering. The working hypothesis is twofold: Reactions that are grouped together are expected to belong to the same pathways, or are regulated in a similar manner (e.g., by the same allosteric regulator). Furthermore, groups of reactions exhibiting mostly negative correlation towards other reactions indicate different patterns of usage over time and are probably stress induced. To further investigate this hypothesis we cluster the fractional appearance profiles of the previously selected reactions (cf. Materials and Methods). The results of the clustering are shown in Figure 3 (a full list of all clustered reaction names can be found in the Additional file 1: Table S2). The shape of the profile in each cluster suggests two groups of reactions: those which are active across all time points, represented by flat profiles, and those whose usage changes during the progression of stress application, exhibiting fluctuating patterns. More specifically, a reaction which exhibits fractional appearance Canagliflozin greater than zero over all time points is considered to have a flat profile. In contrast, a reaction which exhibits fractional appearance of zero in at least one time point is considered to have a fluctuating pattern. Depending on the employed clustering method, there could be clusters exclusively composed of reactions showing flat or fluctuating profiles as well as clusters containing reactions of both profiles. For instance, for the clustering of fractional appearance profiles under cold stress in Figure 3, we observe that clusters 1, 2, 4, and 6 consist only of reactions with fluctuating profiles, clusters 7 and 9 of reactions with flat profiles, while clusters 3, 5, and 8 include reactions of both types of profiles.Flat patterns represent indispensable metabolic reactionsFor ease of interpretation, we focus our analysis on the 50 reactions of highest fractional appearance for each time-point. The union of these selected reactions over all time points contains 71 and 76 reactions for the cold and heat shock, respectively. Out of these, 43 are conserved between the two stresses. To gain general insights into the patterns of the fractional appearance profiles of these selected reactions, we determined the Kendall correlation (cf. Additional file 1). The value captures (dis)similarities in the temporal usageWe first focus our analysis on reactions whose occurrence in EFMs does not change as a result of perturbation, i.e., on clusters in which all fractional appearance profiles are flat. Those reactions are grouped in cluster 7 and 9 in both cold and heat stress (Figure 3). In total, these clusters consist of 36 and 47 reactions for cold and heat stress, respectively, of which 19 of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 these reactions are conserved between the two stresses. These reactions appear in 5 to 40.

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