BACKGROUND
for complex systems such as biological networks, rather than a precise description of the dynamics, which requires a quantity of kinetic details rarely accessible in large scale systems, it is often more reasonable to use a minimal representation, such as a graph of interactions between the molecular variables of interest  <cit>  and perhaps a sign describing the mode of the pairwise interaction.
such graphical approaches have been extensively used in recent years to model transcriptional  <cit>  and signaling networks  <cit> .
apart from biological systems, signed adjacency graphs have been investigated in several different contexts, such as economics  <cit> , social balance  <cit> , and in the theory of frustrated spin systems  <cit> , see  <cit>  for a survey.
in spite of the minimal amount of information it contains, a signed graph can already be used to study dynamical systems properties.
among the various approaches that have been used for this scope, we recall for example the characterizations of multistationarity of  <cit> , stability  <cit> , and the boolean network analysis of e.g.
<cit> .
in particular, in  <cit>  signed graphs are linked to the theory of monotone dynamical systems  <cit>  and the latter is used as a paradigm to explain the highly predictable and ordered response of biological systems to perturbations.
in a biological network, a response to a perturbation propagating incoherently through the network may result in an unpredictable or contradictory behavior of the system, see example in fig.
<dig>  when its dynamics are always free from such contradictory responses then the system is said monotone  <cit> , see methods for a more rigorous definition.
in dynamical systems language, a monotone system exhibits an ordered response because it lacks sustained oscillations and chaotic behavior, thereby rendering the behavior of the system particularly simple.
hence the investigation of how close a biological system is to being monotone has been the subject of intense research in recent years  <cit> .
from a statistical physics perspective, the problem of determining monotonicity  is equivalent to checking when an ising model with signed interactions has no  frustration  <cit> .
in terms of the signed graph, frustration corresponds to undirected cycles having an odd number of negative edges  <cit> .
see also  <cit>  for another recent use of ising models in the context of complex networks.
in this work we are interested in computing the frustration of biological networks of various types: transcriptional, signaling and metabolic.
when modeling these different classes of networks as signed graphs, we have to use different levels of resolution: for signaling and metabolic networks we start from a set of stoichiometric reactions and obtain the signed graph by taking the signature of the jacobian of the corresponding reaction kinetics, hence an edge represents the contribution of a molecular specie to a kinetic reaction, see  <cit>  and the methods section.
for transcriptional networks, on the contrary, we model interactions at functional level, i.e., we take an edge to represent the entire action of activation/inhibition of a transcription factor on a target gene, and in doing so we lump together many important molecular steps, from the binding of the transcription factor to the promoter region of a target gene to the final release of the newly synthetized mrna molecule.
energetically, such complex process is various  times more relevant than a signaling event or a metabolic reaction.
also the corresponding time scales differ by several orders of magnitude  <cit> .
of course, we are forced to use this coarser level of resolution because the stoichiometric details are different for different transcriptional actions, and are not known systematically .
notice that a similar functional representation, oriented at capturing the "information flow" rather than the "mass flow", is possible also for signaling networks  <cit> .
although it may elucidate better the causal transfer of "information" along the pathways, it seems less appropriate to describe the energetic content of the biochemical transformations necessary for the propagation of the signal than the stoichiometric level which we use in this paper, see supplementary notes in additional file  <dig> for a more detailed discussion.
in any case, the qualitative difference in the modeling assumptions made should always be kept in mind, and the classes of networks analyzed should be connotated accordingly as "transcriptional, at functional level" and "signaling/metabolic, at stoichiometric level".
under these assumptions, the frustration index we observe varies considerably according to the type of network analyzed: it is very low for gene regulatory, networks and much higher for signaling and metabolic networks.
in this paper we propose an interpretation of this different behavior based on the characteristic "energy" associated to the interactions of a graph.
we assume that the costs of the interactions  are all comparable on each class of networks, but not across classes of networks.
in particular, transcriptional edges have a much higher cost than the other classes of interactions, and we can speculate that on an evolutionary scale this may have strongly disfavored the development of interactions leading to frustration, i.e., of incoherent or contradictory transcriptional orders.
for the "cheaper" signaling and metabolic interactions, instead, such a tight control may not be required, especially since a higher frustration may induce a richer and more complex dynamical behavior.
we know from the theory of ising models that it is energetically favorable for neighbouring spins to be aligned when the interaction constant is positive and to be antialigned when it is negative.
if we associate to the frustration index the global optimum of an "energy" function describing the amount of such unsatisfied interactions, then we can say that networks with low frustration will have a "ground state"  of lower energy than more frustrated networks.
in addition, rather than just focusing on the energy of the optimal configuration, we can average the state of the system over all possible perturbations, and study what is the average frustration of a network.
in particular, then, if we take the strength of the interactions of a network as "cooling" parameter, we can use statistical physics arguments  <cit>  to describe how the probability of occupancy of the global minimum of the energy varies with the interaction strength, and therefore how monotonically a network behaves in average in response to random perturbations.
what we observe is that the more frustrated signaling/metabolic networks achieve "order"  in a range of interaction energies which is lower than for the transcriptional networks, meaning that these networks  tend to respond to perturbations as coherently as they can even for moderate values of energy.
this behavior partially compensates for the higher frustration, which, as already mentioned, might be instrumental to the achievement of more complex dynamics than those required for the transcriptional networks.
the transcriptional networks, on the other hand, only contain strong interactions and are therefore not concerned with the lower energetic regime.
coherently, they show a topological structure richer in tree-like subgraphs, which disfavor the transition to ordered behavior, and which are absent in the other classes of networks.
that signaling and metabolic networks may require a lower energetic content to experience a transition to ordered behavior is also confirmed by the structure of their energy landscapes which, unlike for the transcriptional networks, lack high and neat energetic barriers around the global optima, meaning that reconfiguration to the ground state can be easily achieved even at modest energies.
RESULTS
the representation of a biological network as an n-dimensional signed adjacency matrix is given by a matrix  of elements jij ∈ {± <dig>  0}, i, j =  <dig> ..., n.  is assumed symmetric , and with zero diagonal , see  <cit>  and the methods section for details on the formulation of the problem.
as explained in the methods, for a stoichiometric system we can assume that  corresponds to the signature of the jacobian linearization around an equilibrium point.
for networks represented as functional activatory/inhibitory actions, the interpretation is even more straightforward.
coherently with our choice of model, we assume that also the perturbations affecting the system around its equilibrium point are of unit magnitude in each component, si ∈ {±1}, i =  <dig> ..., n. in correspondence of a vector s = t of such signed perturbations, or "spin" variables, let us consider the "energy" function
which expresses the total cost associated to the perturbation s. assuming that all interactions of a network have the same strength, |jij| =  <dig> whenever jij ≠  <dig>  the cost of each interaction depends on the sign of each nonzero jij: for jij >  <dig>  the aligned si, sj spin configuration is more energetically favorable  than the antialigned one  and viceversa for jij <  <dig>  of all 2n possible spin assignments, those respecting monotonicity will be such that jijsisi >  <dig> on each edge of the graph, i.e., those contributing to minimizing h. a spin system is said frustrated when not all these constraints jijsisj >  <dig> can be satisfied simultaneously by any assignment.
computing how far a given network is from being monotone corresponds to computing the ground state sground, i.e., the spin assignment that globally minimizes .
it has been shown  <cit>  that this is an np-hard problem, equivalent to the max-cut problem or, in terms of the ising model, to computing the exact frustration index of the network  <cit> , call it δ. in  <cit>  , we proposed efficient heuristic algorithms providing fairly tight upper and lower bounds for δ in biological networks of the size of the thousands nodes.
from the theory of monotone systems ,  is monotone if and only if there exists a diagonal signature matrix dσ  such that  has all nonnegative entries, see lemma  <dig>  in  <cit> .
σ and  have different sign patterns but the same frustration index δ, as dσ is a change of sign through a cut set of the graph of  and such "gauge transformations" dσ  <cit>  leave the sign of each cycle of the graph  unaltered.
let us consider first as an illustrative example the yeast cell cycle network introduced in  <cit>  in the context of boolean networks, see fig.
<dig>  with respect to the original graph of  <cit> , we drop the self-loops and consider the underlying undirected graph .
the number of negative signs on the symmetrized adjacency matrix  is  <dig>  however, a gauge transformation on the three nodes cib <dig>  clb <dig>  and cln <dig>  yields a σ with only  <dig> negative edges, which is a global optimum for the frustration index δ, see fig.
<dig> .
the presence of frustrated cycles in a network leads to a lack of coherence in the response of the system to perturbations.
this can be observed in fig.
<dig>  where the response of the yeast cell cycle and of a monotone network built on the same graph are compared.
the behavior of the non-monotone cell cycle network is less predictable and potentially contradictory .
it is then important to have an estimate of how close a true network is to being monotone i.e., frustration-free.
our algorithms allow to obtain a σ with as low as possible number of negative signs also for large-scale networks.
this number is typically close to δ, meaning that it is now much easier to localize on the graph of σ the potentially frustrated edges .
another consequence is that the candidate ground state for σ that globally minimizes  is now straightforward to identify, as it corresponds to the "all spins up" configuration, call it  <dig>  hence, the candidate ground state for the original  can be found reversing the gauge transformation: sground = dσ  <dig>  approximate values for the frustration index δ and for the corresponding energy minimum  not very far from the true ones can therefore be computed.
frustration in large-scale biological networks
for the eight large-scale biological networks listed in table  <dig>  four transcriptional , two signaling  and two metabolic , we considered the corresponding signed graphs  and estimated δ through the algorithms mentioned above, see table  <dig>  the theory of signed graphs provides us with a theoretical upper bound on the frustration index , call it δmax, which is a function only of the number of nodes, edges and cycles of the networks.
the ratio δ/δmax, fig.
<dig>  shows a marked difference between transcriptional networks and signaling/metabolic networks, with the former exhibiting a consistently lower level of frustration than the latter.
the upper bound δmax, however, disregards completely the topological structure and the sign arrangements of a network.
to take into account also these parameters, we constructed a null-model of the networks, obtained by randomly reshuffling the signs of the edges, while maintaining the same number of positive and negative edges of the original graph, see supplementary notes in additional file  <dig> for more details.
for the z-score of this null model, a negative value means that the edges are arranged in order to decrease frustration.
we can observe in fig.
<dig> that all the transcriptional networks have a negative z-score, and only them .
the characteristic property of the transcriptional networks that enhances monotonicity is the tendency of many nodes to have a skewed distribution of signs in their edges, see fig.
<dig>  up to a gauge transformation, in fact, highly asymmetric sign distributions correspond to highly positive sign concentrations, hence closer to monotone than random sign distributions.
the "packing" of signs on certain nodes is primarily due to the mode of action of the transcription factors.
although dual role  transcription factors exist in both prokaryotes and eukaryotes  <cit> , most transcription factors seem to be playing only one role on their target genes.
the nature of this single role is sometimes associated to the regulatory domains found on the proteins, especially for activator domains, which are usually enriched in proline, glutamine or acidic amino acid residues  <cit> .
the dual role transcription factors are usually able to perform opposite functions according to possible different positions of their binding sequence with respect to the gene sequence, or according to different cellular contexts, or simply enhancing only the formation of the closed complex dna-rna polymerase  <cit> .
for example, 71% of the e.coli transcription factors function only as activators or repressors, fig.
<dig>  the ontological analysis of the dual role transcription factors is significantly enriched for categories such as interfacing the cell with its extracellular environment and for the elaboration of external stimuli .
hence mixed role transcription factors are more often mediating signaling events than their single role counterparts.
it is shown in fig.
<dig> that all transcriptional networks  have sign arrangements on the edges that are more skewed than expected  and also this property contributes to their monotonicity .
another structural difference between transcriptional and signaling/metabolic networks is the overrepresentation in these last classes of short frustrated cycles.
as explained in the supplementary notes in additional file  <dig>  this characteristic is encoded in the level of detail  that we choose to represent our networks, and expresses the lack of global monotonicity of a biochemical reaction involving multiple reagents, see also  <cit> .
n and m are the number of nodes and edges of the directed graph; "leaves" is the number of nodes not involved in any undirected cycle.
more details on the networks are provided in tables s1-s <dig>
δlow and δup are the computational lower and upper bounds found for δ by the algorithms described in supplementary notes in additional file 1; δmax is the theoretical upper bound.
the z-score statistics for the frustration index δ is based on the null model obtained reshuffling the signs .
δnull and δnull are mean and standard deviation of the null model.
the z-score compares this statistics with the "true" δ .
average frustration and ordered response
the values of δ and h alone are not enough to characterize how monotonically the system behaves in average.
in fact, the energy landscape of frustrated ising spin systems is known to be usually rugged  <cit> , and the presence of a single deep minimum in  is not enough to guarantee that the energy averaged over all configurations s  is indeed more negative than in other systems whose energy landscape is characterized by valleys which are maybe less deep but with larger basins.
in other words, to characterize how monotone is the response of the system to arbitrarily complex perturbations we have to consider the average value that h assumes over all possible spin assignments, weighted by the probability of each s. this "internal energy", call it ⟨h⟩, is an indicator of how coherently the system is behaving in average: the more negative ⟨h⟩ is, the less the responses of the system to perturbations are "contradictory" at some fan-in node or along directed cycles.
denote with  the partition function of the system, β ∈ ℝ+.
as usual in statistical physics, the partition function z is the normalization factor that renders the frequencies of the various spin states true probability densities.
for spin systems, β has the meaning of an inverse temperature and it is normally used as "cooling" parameter, i.e., when β→ ∞ the probability of the state s, p, tends to concentrate on the ground states: p →  <dig> as β → ∞.
in the context of biological networks, the temperature is taken as ~  <dig> k and it is not a varying parameter.
however, we can use β to describe the strength of the interactions of a network.
recall that in forming the energy ,  was taken as a signed adjacency matrix with all interactions equal to  <dig>  regardless of the nature of the network studied.
as a matter of fact, metabolic, signaling and transcriptional interactions are characterized by widely different energetic costs.
in particular, if in our stoichiometric representation a metabolic reaction or a signaling event might have a comparable energetic content, a link in a gene regulatory network describes the entire cascade of events in which the transcription of a gene can be broken down and overall its cost is much higher than in the other networks.
hence, in our fixed temperature context, taking into account the interaction cost β rescales h to the "absolute" energy βh.
the probability of a given configuration s, p = e-βhz- <dig>  is a function of β and is maximized in the  ground state sground.
as for spin systems, , i.e., when β is large enough, in average the system will always be found in the configuration sground which minimizes the energy  and which exhibits the least frustration for the network.
using β as a lagrange multiplier, the internal energy ⟨h⟩ is defined as the expectation value of h,
and expresses this simultaneous weighting of the configurations by their degeneracy and energetic content.
the more negative ⟨h⟩ is, the more we expect the system to respond coherently to a generic perturbation.
for any β >  <dig>  ⟨h⟩ <  <dig> and, as β increases, ⟨h⟩ reaches a stationary value, see fig.
<dig>  for spin systems, small values of β represent a regimen where thermal fluctuations are dominant and all states tend to be equally populated.
as β increases, a spin system usually undergoes a phase transition characterized by the appearance of long range correlations in the expectation values assumed by the si.
for our biological networks, when β  is too small, the behavior of the network tends to be random  regardless of the monotonicity of the network, a clear obstacle to carrying out any meaningful task.
on the other hand, when β → ∞, the probability concentrates exclusively on the ground states  becomes a dirac delta function) and the behavior of the system becomes as ordered as its frustration index allows, i.e., the system response is as coherent and coordinated as possible, regardless of the type of perturbation, see fig.
<dig> .
it is then important to see how the probabilities of the various states p and the internal energy  vary as a function of β on the various categories of networks under exam.
computing p and ⟨h⟩ exactly is impossible for systems larger than a few tens of nodes.
for larger networks we shall make use of a mean field approximation for heterogeneous networks  <cit> .
this approximation, see the "methods" section for details, allows to estimate the expectation value ⟨sσ⟩ in the gauge transformed basis, and the corresponding mean field energy hmf.
fig.
<dig> shows the behavior of ⟨sσ⟩ and hmf for a transcriptional, a signaling and a metabolic network as function of β. in all three cases, the population concentrates in the ground state when β grows, and, correspondingly, hmf achieves its global minimum.
the true characteristic value of the interaction strength β at which each of the classes of networks should be computed is unknown, except for the suggestion that βtranscr ≫ βsignal ~ βmetab.
interestingly, as β grows, the transcriptional network is slower to reach its energetic minimum than the other two networks, and likewise for the other  <dig> networks, see table s <dig> and fig.
s <dig>  this shift of the coherence barrier towards the low energetic regions is a consequence of the topology of the networks.
in fact, as can be seen on fig.
<dig>  also the completely monotone networks built on the same graphs  as well as other networks with random sign assignment to the same edges as our   present the same characteristic patterns in spite of different δ. a feature behind this difference is the already mentioned overrepresentation of closed undirected cycles of short length in the structure of metabolic and signaling networks.
also the lower dispersion in the number of connectivity degree classes k in these networks contributes to the quick convergence of ⟨sσ⟩ to  <dig>  however, the main reason behind the different thresholds for β is the presence or less of leaves in the graph.
for example, the e.coli transcriptional network has 38% of the nodes that are not involved in any  cycle, see table  <dig>  dropping these nodes and concentrating on the 2-core of the undirected graph, we obtain mean field plots in which the threshold for order is lower, and similar to those of the signaling/metabolic networks, see fig.
s <dig>  all of our transcriptional networks have a high percentage of nodes that are leaves, much higher that the signaling/metabolic networks, see table  <dig>  the complete lack of feedback, characteristic of tree-like subnetworks, disfavours the achievement of a globally ordered behavior, which is more easily achieved when short cycles, like the 3-node motifs of signaling/metabolic networks, are abundant.
this is expected from the theory of spin systems, where long-range correlations are more easily obtained in dense graphs than in sparse ones.
of course adding leaves to a graph does not change its monotonicity properties .
the qualitative difference in the phase transition to order between transcriptional and signaling/metabolic networks suggests an interpretation coherent with the different energetic content associated to the classes of networks.
in fact, we can say that since βtranscr is high, it is much less plausible for a transcriptional network to be operating in a regimen of low β than it is for signaling/metabolic networks.
on the contrary, for these last two classes of networks, it is not unlikely to have interactions of medium-low strength.
hence it gets much more important that ⟨sσ⟩ →  <dig> even in correspondence of moderate values of β, because this helps in maintaining a coherent behavior in response to perturbations, as required in order to carry out correctly a biological task.
sampling the energy landscape
further information, from a different perspective, can be obtained studying the structure of the energy landscape of the different networks  <cit> .
in order to have a picture of how this landscape looks like, we have applied our frustration minimization algorithms to uniformly distributed initial conditions and registered the local and global minima achieved in the process .
fig.
<dig> shows these distributions of minima as a function of the relative hamming distance.
for the transcriptional network of e.coli and the yeast metabolic network, the global minima are all localized in a small region, while egfr has two broader valleys of global minima.
in all three cases, the global minima are surrounded by many local minima, thus confirming the ruggedness of the landscapes.
as can be seen on fig.
s <dig>  unlike egfr and the metabolic network, the local minima of the transcriptional network of e.coli tend to have an energetic difference from the global ones which grows linearly with the distance.
in addition, the separation between the well of global minima and its surroundings is much more neat in e.coli than in the other two networks, as can be seen on the montecarlo trajectories of fig.
<dig> and even more clearly on the average gradient of h .
see also figs.
s <dig>  s <dig>  and s <dig> for analogous consideration on the remaining  <dig> networks.
overall, it appears that global and local minima in the transcriptional networks are separated by high and steep energetic barriers, while on the other networks there always exist low-energy routes between random spin configurations and global minima, possibly passing through low-energy local minima.
this of course facilitates the achievement of the ground state and the creation of global order even in a regime of moderate values of β.
discussion
for a gene regulatory network, an edge represents the cost of the entire action of transcription of a gene.
this is a complex, multistep process: for prokaryotes, for example, it includes the binding of the transcription factor to the dna, the recruitment of a polymerase, the unwinding of the dna helix, the detachment of the σ -factor and the conformational changes in the polymerase preceeding elongation, the release of both the dna and of the complete mrna at the termination phase.
the energetic cost and time constant of such a complex process are relevant for a cell.
hence, especially in lower organisms, it is natural to expect that in a transcriptional network the genes behave in concert and that the fraction of the gene-gene interactions that contribute to minimizing the energy in response to perturbations is substantially larger than in a metabolic or signaling network, as a frustrated bond costs much more to the cell and its effect lasts much longer.
in particular, frustrations manifest themselves on the cycles of the underlying undirected graph of the network as contradictory transcriptional orders.
while changing the transcriptional commands is necessary to cope with e.g.
different environmental conditions, encoding them as frustrated cycles can easily lead to unpredictable or erroneous dynamical behavior.
therefore, in spite of the presence of certain characteristic motifs leading to frustration , overall the transcriptional networks we analyze are indeed near-monotone.
both the topology and the sign assignments to the nodes of the transcriptional networks contribute to achieve a degree of monotonicity which is higher than expected from null models.
on the contrary, incoherent signaling or metabolic actions are energetically much less relevant than a single transcriptional event and can be easily tolerated by the cell, especially since nonmonotone patterns favour a richer dynamical behavior.
while the level of detail at which we model our networks  certainly contributes to the systematic differences in the frustration index, other factors such as the tendency of the transcriptional networks to have skewed sign distributions are also crucial in attaining a low frustration.
it is interesting, then, to notice that in e.coli the transcription factors violating this rule are primarily involved in the mediation of external signaling, rather than in regulatory or structural functions .
for spin systems, the tendency to satisfy pairwise all interactions grows when the temperature decreases, although in a frustrated ising spin system all the conditions can never be satisfied simultaneously.
in this paper, we consider the strength of the interactions as the key factor that determines the increase in the probability of finding the system in its ground state .
if we parametrize the networks by the interaction strength and study the probability of finding the system response in the ground state as a function of this cost, we observe that for signaling/metabolic networks it is higher than for transcriptional networks in the region of medium/low values of the interactions.
this behavior, which is due to the topological structure of the networks and to the energy landscape it determines, could reflect the tendency of signaling/metabolic networks to attain a globally ordered response in spite of the weaker energetic content of their interactions.
as such, it helps maintaining coherence of the response in spite of the higher level of frustration of these networks .
for transcriptional networks, on the other hand, owing to the strong interactions, the regime of low energies is less important, hence tree-like motifs, which hinder the establishment of long-range correlations, are abundant.
a montecarlo investigation of the energy landscape of the networks  <cit>  suggests that transcriptional networks tend to have a more funneled landscape than the other networks , with a single deep well of global minima delimited by high barriers, while in signaling and metabolic networks the optima are surrounded by local minima of comparable energy.
order in these classes of networks is favored also by the lack of neat energetic barriers separating local and global optima, which enables the reconfiguration to the global optimum through low-energy paths.
several are the caveat and limitations of our study.
first of all, the different levels of resolution for the different classes of networks may be a source  of the systematic differences we are observing.
hints in this direction come for example from the observation that networks at functional level tend to have less cycles than networks at stoichiometric level , and that functional models of signaling pathways may also have asymmetric sign distributions .
this is observed to some extent in the functional model of the hippocampal signaling network proposed in  <cit> .
notice that this network has a large fraction  of interactions representing protein-protein or protein-ligand bindings, to which it is unclear how to associate a sign in an unambiguous manner.
the ambiguity of course also propagates to the level of frustration one obtains correspondingly.
more generally, we are not aware of any systematic way to map the pathway charts available at stoichiometric level to the functional level, allowing to univocally assign a sign to each edge without at the same time loosing in this process a large part of the molecular species involved.
notice also that the opposite option, namely representing transcriptional networks at stoichiometric level, is de facto impossible with our current knowledge.
another important source of uncertainty comes from the limited coverage of the biological networks currently available.
in particular, for the transcriptional networks, the fraction of target genes having at least a transcription factor is below 50% of the genes.
furthermore, our considerations about an higher than expected monotonicity may very well be overturned once more complex organisms  are taken into account.
CONCLUSIONS
in conclusion, we have observed that distinct classes of biological networks seem to be characterizable by different features in response to perturbations.
at least when we model transcriptional networks at functional level  and signaling and metabolic networks at stoichiometric level, we can observe that transcriptional networks appear to be less frustrated than expected and much less frustrated than signaling and metabolic networks, meaning that they might admit highly coherent responses to perturbations.
on the other hand, the signaling/metabolic networks seem to have the ability to achieve an average ordered response in a lower range of interaction strengths than the transcriptional networks.
we explain the first feature as the need to avoid as much as possible erroneous or contradictory transcriptional actions which would cost much more to the cell than analogous incoherent signaling/metabolic events.
the second feature may partially compensate for the higher frustration of these last networks, by lowering the interaction strength needed for a transition to ordered response , and thereby ensuring the effectiveness of this reduced coherent behavior in an energetic range more critical for these classes of networks.

 for complex systems such as biological networks, rather than a precise description of the dynamics, which requires a quantity of kinetic details rarely accessible in large scale systems, it is often more reasonable to use a minimal representation, such as a graph of interactions between the molecular variables of interest and perhaps a sign describing the mode of the pairwise interaction. such graphical approaches have been extensively used in recent years to model transcriptional and signaling networks. apart from biological systems, signed adjacency graphs have been investigated in several different contexts, such as economics, social balance, and in the theory of frustrated spin systems, see for a survey. in spite of the minimal amount of information it contains, a signed graph can already be used to study dynamical systems properties. among the various approaches that have been used for this scope, we recall for example the characterizations of multistationarity of, stability, and the boolean network analysis of e.g.. in particular, in signed graphs are linked to the theory of monotone dynamical systems and the latter is used as a paradigm to explain the highly predictable and ordered response of biological systems to perturbations. in a biological network, a response to a perturbation propagating incoherently through the network may result in an unpredictable or contradictory behavior of the system, see example in fig. when its dynamics are always free from such contradictory responses then the system is said monotone, see methods for a more rigorous definition. in dynamical systems language, a monotone system exhibits an ordered response because it lacks sustained oscillations and chaotic behavior, thereby rendering the behavior of the system particularly simple. hence the investigation of how close a biological system is to being monotone has been the subject of intense research in recent years. from a statistical physics perspective, the problem of determining monotonicity is equivalent to checking when an ising model with signed interactions has no frustration. in terms of the signed graph, frustration corresponds to undirected cycles having an odd number of negative edges. see also for another recent use of ising models in the context of complex networks. in this work we are interested in computing the frustration of biological networks of various types: transcriptional, signaling and metabolic. when modeling these different classes of networks as signed graphs, we have to use different levels of resolution: for signaling and metabolic networks we start from a set of stoichiometric reactions and obtain the signed graph by taking the signature of the jacobian of the corresponding reaction kinetics, hence an edge represents the contribution of a molecular specie to a kinetic reaction, see and the methods section. for transcriptional networks, on the contrary, we model interactions at functional level, i.e., we take an edge to represent the entire action of activation/inhibition of a transcription factor on a target gene, and in doing so we lump together many important molecular steps, from the binding of the transcription factor to the promoter region of a target gene to the final release of the newly synthetized mrna molecule. energetically, such complex process is various times more relevant than a signaling event or a metabolic reaction. also the corresponding time scales differ by several orders of magnitude. of course, we are forced to use this coarser level of resolution because the stoichiometric details are different for different transcriptional actions, and are not known systematically. notice that a similar functional representation, oriented at capturing the "information flow" rather than the "mass flow", is possible also for signaling networks. although it may elucidate better the causal transfer of "information" along the pathways, it seems less appropriate to describe the energetic content of the biochemical transformations necessary for the propagation of the signal than the stoichiometric level which we use in this paper, see supplementary notes in additional file for a more detailed discussion. in any case, the qualitative difference in the modeling assumptions made should always be kept in mind, and the classes of networks analyzed should be connotated accordingly as "transcriptional, at functional level" and "signaling/metabolic, at stoichiometric level". under these assumptions, the frustration index we observe varies considerably according to the type of network analyzed: it is very low for gene regulatory, networks and much higher for signaling and metabolic networks. in this paper we propose an interpretation of this different behavior based on the characteristic "energy" associated to the interactions of a graph. we assume that the costs of the interactions are all comparable on each class of networks, but not across classes of networks. in particular, transcriptional edges have a much higher cost than the other classes of interactions, and we can speculate that on an evolutionary scale this may have strongly disfavored the development of interactions leading to frustration, i.e., of incoherent or contradictory transcriptional orders. for the "cheaper" signaling and metabolic interactions, instead, such a tight control may not be required, especially since a higher frustration may induce a richer and more complex dynamical behavior. we know from the theory of ising models that it is energetically favorable for neighbouring spins to be aligned when the interaction constant is positive and to be antialigned when it is negative. if we associate to the frustration index the global optimum of an "energy" function describing the amount of such unsatisfied interactions, then we can say that networks with low frustration will have a "ground state" of lower energy than more frustrated networks. in addition, rather than just focusing on the energy of the optimal configuration, we can average the state of the system over all possible perturbations, and study what is the average frustration of a network. in particular, then, if we take the strength of the interactions of a network as "cooling" parameter, we can use statistical physics arguments to describe how the probability of occupancy of the global minimum of the energy varies with the interaction strength, and therefore how monotonically a network behaves in average in response to random perturbations. what we observe is that the more frustrated signaling/metabolic networks achieve "order" in a range of interaction energies which is lower than for the transcriptional networks, meaning that these networks tend to respond to perturbations as coherently as they can even for moderate values of energy. this behavior partially compensates for the higher frustration, which, as already mentioned, might be instrumental to the achievement of more complex dynamics than those required for the transcriptional networks. the transcriptional networks, on the other hand, only contain strong interactions and are therefore not concerned with the lower energetic regime. coherently, they show a topological structure richer in tree-like subgraphs, which disfavor the transition to ordered behavior, and which are absent in the other classes of networks. that signaling and metabolic networks may require a lower energetic content to experience a transition to ordered behavior is also confirmed by the structure of their energy landscapes which, unlike for the transcriptional networks, lack high and neat energetic barriers around the global optima, meaning that reconfiguration to the ground state can be easily achieved even at modest energies.  the representation of a biological network as an n-dimensional signed adjacency matrix is given by a matrix of elements jij ∈ {± 0}, i, j =..., n. is assumed symmetric, and with zero diagonal, see and the methods section for details on the formulation of the problem. as explained in the methods, for a stoichiometric system we can assume that corresponds to the signature of the jacobian linearization around an equilibrium point. for networks represented as functional activatory/inhibitory actions, the interpretation is even more straightforward. coherently with our choice of model, we assume that also the perturbations affecting the system around its equilibrium point are of unit magnitude in each component, si ∈ {±1}, i =..., n. in correspondence of a vector s = t of such signed perturbations, or "spin" variables, let us consider the "energy" function which expresses the total cost associated to the perturbation s. assuming that all interactions of a network have the same strength, |jij| = whenever jij ≠ the cost of each interaction depends on the sign of each nonzero jij: for jij > the aligned si, sj spin configuration is more energetically favorable than the antialigned one and viceversa for jij < of all 2n possible spin assignments, those respecting monotonicity will be such that jijsisi > on each edge of the graph, i.e., those contributing to minimizing h. a spin system is said frustrated when not all these constraints jijsisj > can be satisfied simultaneously by any assignment. computing how far a given network is from being monotone corresponds to computing the ground state sground, i.e., the spin assignment that globally minimizes. it has been shown that this is an np-hard problem, equivalent to the max-cut problem or, in terms of the ising model, to computing the exact frustration index of the network, call it δ. in, we proposed efficient heuristic algorithms providing fairly tight upper and lower bounds for δ in biological networks of the size of the thousands nodes. from the theory of monotone systems, is monotone if and only if there exists a diagonal signature matrix dσ such that has all nonnegative entries, see lemma in. σ and have different sign patterns but the same frustration index δ, as dσ is a change of sign through a cut set of the graph of and such "gauge transformations" dσ leave the sign of each cycle of the graph unaltered. let us consider first as an illustrative example the yeast cell cycle network introduced in in the context of boolean networks, see fig. with respect to the original graph of, we drop the self-loops and consider the underlying undirected graph. the number of negative signs on the symmetrized adjacency matrix is however, a gauge transformation on the three nodes cib clb and cln yields a σ with only negative edges, which is a global optimum for the frustration index δ, see fig.. the presence of frustrated cycles in a network leads to a lack of coherence in the response of the system to perturbations. this can be observed in fig. where the response of the yeast cell cycle and of a monotone network built on the same graph are compared. the behavior of the non-monotone cell cycle network is less predictable and potentially contradictory. it is then important to have an estimate of how close a true network is to being monotone i.e., frustration-free. our algorithms allow to obtain a σ with as low as possible number of negative signs also for large-scale networks. this number is typically close to δ, meaning that it is now much easier to localize on the graph of σ the potentially frustrated edges. another consequence is that the candidate ground state for σ that globally minimizes is now straightforward to identify, as it corresponds to the "all spins up" configuration, call it hence, the candidate ground state for the original can be found reversing the gauge transformation: sground = dσ approximate values for the frustration index δ and for the corresponding energy minimum not very far from the true ones can therefore be computed. frustration in large-scale biological networks for the eight large-scale biological networks listed in table four transcriptional, two signaling and two metabolic, we considered the corresponding signed graphs and estimated δ through the algorithms mentioned above, see table the theory of signed graphs provides us with a theoretical upper bound on the frustration index, call it δmax, which is a function only of the number of nodes, edges and cycles of the networks. the ratio δ/δmax, fig. shows a marked difference between transcriptional networks and signaling/metabolic networks, with the former exhibiting a consistently lower level of frustration than the latter. the upper bound δmax, however, disregards completely the topological structure and the sign arrangements of a network. to take into account also these parameters, we constructed a null-model of the networks, obtained by randomly reshuffling the signs of the edges, while maintaining the same number of positive and negative edges of the original graph, see supplementary notes in additional file for more details. for the z-score of this null model, a negative value means that the edges are arranged in order to decrease frustration. we can observe in fig. that all the transcriptional networks have a negative z-score, and only them. the characteristic property of the transcriptional networks that enhances monotonicity is the tendency of many nodes to have a skewed distribution of signs in their edges, see fig. up to a gauge transformation, in fact, highly asymmetric sign distributions correspond to highly positive sign concentrations, hence closer to monotone than random sign distributions. the "packing" of signs on certain nodes is primarily due to the mode of action of the transcription factors. although dual role transcription factors exist in both prokaryotes and eukaryotes, most transcription factors seem to be playing only one role on their target genes. the nature of this single role is sometimes associated to the regulatory domains found on the proteins, especially for activator domains, which are usually enriched in proline, glutamine or acidic amino acid residues. the dual role transcription factors are usually able to perform opposite functions according to possible different positions of their binding sequence with respect to the gene sequence, or according to different cellular contexts, or simply enhancing only the formation of the closed complex dna-rna polymerase. for example, 71% of the e.coli transcription factors function only as activators or repressors, fig. the ontological analysis of the dual role transcription factors is significantly enriched for categories such as interfacing the cell with its extracellular environment and for the elaboration of external stimuli. hence mixed role transcription factors are more often mediating signaling events than their single role counterparts. it is shown in fig. that all transcriptional networks have sign arrangements on the edges that are more skewed than expected and also this property contributes to their monotonicity. another structural difference between transcriptional and signaling/metabolic networks is the overrepresentation in these last classes of short frustrated cycles. as explained in the supplementary notes in additional file this characteristic is encoded in the level of detail that we choose to represent our networks, and expresses the lack of global monotonicity of a biochemical reaction involving multiple reagents, see also. n and m are the number of nodes and edges of the directed graph; "leaves" is the number of nodes not involved in any undirected cycle. more details on the networks are provided in tables s1-s δlow and δup are the computational lower and upper bounds found for δ by the algorithms described in supplementary notes in additional file 1; δmax is the theoretical upper bound. the z-score statistics for the frustration index δ is based on the null model obtained reshuffling the signs. δnull and δnull are mean and standard deviation of the null model. the z-score compares this statistics with the "true" δ. average frustration and ordered response the values of δ and h alone are not enough to characterize how monotonically the system behaves in average. in fact, the energy landscape of frustrated ising spin systems is known to be usually rugged, and the presence of a single deep minimum in is not enough to guarantee that the energy averaged over all configurations s is indeed more negative than in other systems whose energy landscape is characterized by valleys which are maybe less deep but with larger basins. in other words, to characterize how monotone is the response of the system to arbitrarily complex perturbations we have to consider the average value that h assumes over all possible spin assignments, weighted by the probability of each s. this "internal energy", call it ⟨h⟩, is an indicator of how coherently the system is behaving in average: the more negative ⟨h⟩ is, the less the responses of the system to perturbations are "contradictory" at some fan-in node or along directed cycles. denote with the partition function of the system, β ∈ ℝ+. as usual in statistical physics, the partition function z is the normalization factor that renders the frequencies of the various spin states true probability densities. for spin systems, β has the meaning of an inverse temperature and it is normally used as "cooling" parameter, i.e., when β→ ∞ the probability of the state s, p, tends to concentrate on the ground states: p → as β → ∞. in the context of biological networks, the temperature is taken as ~ k and it is not a varying parameter. however, we can use β to describe the strength of the interactions of a network. recall that in forming the energy, was taken as a signed adjacency matrix with all interactions equal to regardless of the nature of the network studied. as a matter of fact, metabolic, signaling and transcriptional interactions are characterized by widely different energetic costs. in particular, if in our stoichiometric representation a metabolic reaction or a signaling event might have a comparable energetic content, a link in a gene regulatory network describes the entire cascade of events in which the transcription of a gene can be broken down and overall its cost is much higher than in the other networks. hence, in our fixed temperature context, taking into account the interaction cost β rescales h to the "absolute" energy βh. the probability of a given configuration s, p = e-βhz- is a function of β and is maximized in the ground state sground. as for spin systems,, i.e., when β is large enough, in average the system will always be found in the configuration sground which minimizes the energy and which exhibits the least frustration for the network. using β as a lagrange multiplier, the internal energy ⟨h⟩ is defined as the expectation value of h, and expresses this simultaneous weighting of the configurations by their degeneracy and energetic content. the more negative ⟨h⟩ is, the more we expect the system to respond coherently to a generic perturbation. for any β > ⟨h⟩ < and, as β increases, ⟨h⟩ reaches a stationary value, see fig. for spin systems, small values of β represent a regimen where thermal fluctuations are dominant and all states tend to be equally populated. as β increases, a spin system usually undergoes a phase transition characterized by the appearance of long range correlations in the expectation values assumed by the si. for our biological networks, when β is too small, the behavior of the network tends to be random regardless of the monotonicity of the network, a clear obstacle to carrying out any meaningful task. on the other hand, when β → ∞, the probability concentrates exclusively on the ground states becomes a dirac delta function) and the behavior of the system becomes as ordered as its frustration index allows, i.e., the system response is as coherent and coordinated as possible, regardless of the type of perturbation, see fig.. it is then important to see how the probabilities of the various states p and the internal energy vary as a function of β on the various categories of networks under exam. computing p and ⟨h⟩ exactly is impossible for systems larger than a few tens of nodes. for larger networks we shall make use of a mean field approximation for heterogeneous networks. this approximation, see the "methods" section for details, allows to estimate the expectation value ⟨sσ⟩ in the gauge transformed basis, and the corresponding mean field energy hmf. fig. shows the behavior of ⟨sσ⟩ and hmf for a transcriptional, a signaling and a metabolic network as function of β. in all three cases, the population concentrates in the ground state when β grows, and, correspondingly, hmf achieves its global minimum. the true characteristic value of the interaction strength β at which each of the classes of networks should be computed is unknown, except for the suggestion that βtranscr ≫ βsignal ~ βmetab. interestingly, as β grows, the transcriptional network is slower to reach its energetic minimum than the other two networks, and likewise for the other networks, see table s and fig. s this shift of the coherence barrier towards the low energetic regions is a consequence of the topology of the networks. in fact, as can be seen on fig. also the completely monotone networks built on the same graphs as well as other networks with random sign assignment to the same edges as our present the same characteristic patterns in spite of different δ. a feature behind this difference is the already mentioned overrepresentation of closed undirected cycles of short length in the structure of metabolic and signaling networks. also the lower dispersion in the number of connectivity degree classes k in these networks contributes to the quick convergence of ⟨sσ⟩ to however, the main reason behind the different thresholds for β is the presence or less of leaves in the graph. for example, the e.coli transcriptional network has 38% of the nodes that are not involved in any cycle, see table dropping these nodes and concentrating on the 2-core of the undirected graph, we obtain mean field plots in which the threshold for order is lower, and similar to those of the signaling/metabolic networks, see fig. s all of our transcriptional networks have a high percentage of nodes that are leaves, much higher that the signaling/metabolic networks, see table the complete lack of feedback, characteristic of tree-like subnetworks, disfavours the achievement of a globally ordered behavior, which is more easily achieved when short cycles, like the 3-node motifs of signaling/metabolic networks, are abundant. this is expected from the theory of spin systems, where long-range correlations are more easily obtained in dense graphs than in sparse ones. of course adding leaves to a graph does not change its monotonicity properties. the qualitative difference in the phase transition to order between transcriptional and signaling/metabolic networks suggests an interpretation coherent with the different energetic content associated to the classes of networks. in fact, we can say that since βtranscr is high, it is much less plausible for a transcriptional network to be operating in a regimen of low β than it is for signaling/metabolic networks. on the contrary, for these last two classes of networks, it is not unlikely to have interactions of medium-low strength. hence it gets much more important that ⟨sσ⟩ → even in correspondence of moderate values of β, because this helps in maintaining a coherent behavior in response to perturbations, as required in order to carry out correctly a biological task. sampling the energy landscape further information, from a different perspective, can be obtained studying the structure of the energy landscape of the different networks. in order to have a picture of how this landscape looks like, we have applied our frustration minimization algorithms to uniformly distributed initial conditions and registered the local and global minima achieved in the process. fig. shows these distributions of minima as a function of the relative hamming distance. for the transcriptional network of e.coli and the yeast metabolic network, the global minima are all localized in a small region, while egfr has two broader valleys of global minima. in all three cases, the global minima are surrounded by many local minima, thus confirming the ruggedness of the landscapes. as can be seen on fig. s unlike egfr and the metabolic network, the local minima of the transcriptional network of e.coli tend to have an energetic difference from the global ones which grows linearly with the distance. in addition, the separation between the well of global minima and its surroundings is much more neat in e.coli than in the other two networks, as can be seen on the montecarlo trajectories of fig. and even more clearly on the average gradient of h. see also figs. s s and s for analogous consideration on the remaining networks. overall, it appears that global and local minima in the transcriptional networks are separated by high and steep energetic barriers, while on the other networks there always exist low-energy routes between random spin configurations and global minima, possibly passing through low-energy local minima. this of course facilitates the achievement of the ground state and the creation of global order even in a regime of moderate values of β. discussion for a gene regulatory network, an edge represents the cost of the entire action of transcription of a gene. this is a complex, multistep process: for prokaryotes, for example, it includes the binding of the transcription factor to the dna, the recruitment of a polymerase, the unwinding of the dna helix, the detachment of the σ -factor and the conformational changes in the polymerase preceeding elongation, the release of both the dna and of the complete mrna at the termination phase. the energetic cost and time constant of such a complex process are relevant for a cell. hence, especially in lower organisms, it is natural to expect that in a transcriptional network the genes behave in concert and that the fraction of the gene-gene interactions that contribute to minimizing the energy in response to perturbations is substantially larger than in a metabolic or signaling network, as a frustrated bond costs much more to the cell and its effect lasts much longer. in particular, frustrations manifest themselves on the cycles of the underlying undirected graph of the network as contradictory transcriptional orders. while changing the transcriptional commands is necessary to cope with e.g. different environmental conditions, encoding them as frustrated cycles can easily lead to unpredictable or erroneous dynamical behavior. therefore, in spite of the presence of certain characteristic motifs leading to frustration, overall the transcriptional networks we analyze are indeed near-monotone. both the topology and the sign assignments to the nodes of the transcriptional networks contribute to achieve a degree of monotonicity which is higher than expected from null models. on the contrary, incoherent signaling or metabolic actions are energetically much less relevant than a single transcriptional event and can be easily tolerated by the cell, especially since nonmonotone patterns favour a richer dynamical behavior. while the level of detail at which we model our networks certainly contributes to the systematic differences in the frustration index, other factors such as the tendency of the transcriptional networks to have skewed sign distributions are also crucial in attaining a low frustration. it is interesting, then, to notice that in e.coli the transcription factors violating this rule are primarily involved in the mediation of external signaling, rather than in regulatory or structural functions. for spin systems, the tendency to satisfy pairwise all interactions grows when the temperature decreases, although in a frustrated ising spin system all the conditions can never be satisfied simultaneously. in this paper, we consider the strength of the interactions as the key factor that determines the increase in the probability of finding the system in its ground state. if we parametrize the networks by the interaction strength and study the probability of finding the system response in the ground state as a function of this cost, we observe that for signaling/metabolic networks it is higher than for transcriptional networks in the region of medium/low values of the interactions. this behavior, which is due to the topological structure of the networks and to the energy landscape it determines, could reflect the tendency of signaling/metabolic networks to attain a globally ordered response in spite of the weaker energetic content of their interactions. as such, it helps maintaining coherence of the response in spite of the higher level of frustration of these networks. for transcriptional networks, on the other hand, owing to the strong interactions, the regime of low energies is less important, hence tree-like motifs, which hinder the establishment of long-range correlations, are abundant. a montecarlo investigation of the energy landscape of the networks suggests that transcriptional networks tend to have a more funneled landscape than the other networks, with a single deep well of global minima delimited by high barriers, while in signaling and metabolic networks the optima are surrounded by local minima of comparable energy. order in these classes of networks is favored also by the lack of neat energetic barriers separating local and global optima, which enables the reconfiguration to the global optimum through low-energy paths. several are the caveat and limitations of our study. first of all, the different levels of resolution for the different classes of networks may be a source of the systematic differences we are observing. hints in this direction come for example from the observation that networks at functional level tend to have less cycles than networks at stoichiometric level, and that functional models of signaling pathways may also have asymmetric sign distributions. this is observed to some extent in the functional model of the hippocampal signaling network proposed in. notice that this network has a large fraction of interactions representing protein-protein or protein-ligand bindings, to which it is unclear how to associate a sign in an unambiguous manner. the ambiguity of course also propagates to the level of frustration one obtains correspondingly. more generally, we are not aware of any systematic way to map the pathway charts available at stoichiometric level to the functional level, allowing to univocally assign a sign to each edge without at the same time loosing in this process a large part of the molecular species involved. notice also that the opposite option, namely representing transcriptional networks at stoichiometric level, is de facto impossible with our current knowledge. another important source of uncertainty comes from the limited coverage of the biological networks currently available. in particular, for the transcriptional networks, the fraction of target genes having at least a transcription factor is below 50% of the genes. furthermore, our considerations about an higher than expected monotonicity may very well be overturned once more complex organisms are taken into account.  in conclusion, we have observed that distinct classes of biological networks seem to be characterizable by different features in response to perturbations. at least when we model transcriptional networks at functional level and signaling and metabolic networks at stoichiometric level, we can observe that transcriptional networks appear to be less frustrated than expected and much less frustrated than signaling and metabolic networks, meaning that they might admit highly coherent responses to perturbations. on the other hand, the signaling/metabolic networks seem to have the ability to achieve an average ordered response in a lower range of interaction strengths than the transcriptional networks. we explain the first feature as the need to avoid as much as possible erroneous or contradictory transcriptional actions which would cost much more to the cell than analogous incoherent signaling/metabolic events. the second feature may partially compensate for the higher frustration of these last networks, by lowering the interaction strength needed for a transition to ordered response, and thereby ensuring the effectiveness of this reduced coherent behavior in an energetic range more critical for these classes of networks. 
furthermore, an analysis of the energy landscape shows that signaling and metabolic networks lack energetic barriers around their global optima, a property also favouring global order. in conclusion, transcriptional and signaling/metabolic networks appear to have systematic differences in both the index of frustration and the transition to global order. for large-scale biological networks represented as signed graphs, the index of frustration measures how far a network is from a monotone system, i.e., how incoherently the system responds to perturbations. a possible interpretation of this result is in terms of energetic cost of an interaction: an erroneous or contradictory transcriptional action costs much more than a signaling/metabolic error, and therefore must be avoided as much as possible. averaging over all possible perturbations, however, we also find that unlike for transcriptional networks, in the signaling/metabolic networks the probability of finding the system in its least frustrated configuration tends to be high also in correspondence of a moderate energetic regime, meaning that, in spite of the higher frustration, these networks can achieve a globally ordered response to perturbations even for moderate values of the strength of the interactions. in this paper we find that the frustration is systematically lower in transcriptional networks than in signaling and metabolic networks.
 for large-scale biological networks represented as signed graphs, the index of frustration measures how far a network is from a monotone system, i.e., how incoherently the system responds to perturbations.  in this paper we find that the frustration is systematically lower in transcriptional networks than in signaling and metabolic networks. a possible interpretation of this result is in terms of energetic cost of an interaction: an erroneous or contradictory transcriptional action costs much more than a signaling/metabolic error, and therefore must be avoided as much as possible. averaging over all possible perturbations, however, we also find that unlike for transcriptional networks, in the signaling/metabolic networks the probability of finding the system in its least frustrated configuration tends to be high also in correspondence of a moderate energetic regime, meaning that, in spite of the higher frustration, these networks can achieve a globally ordered response to perturbations even for moderate values of the strength of the interactions. furthermore, an analysis of the energy landscape shows that signaling and metabolic networks lack energetic barriers around their global optima, a property also favouring global order.  in conclusion, transcriptional and signaling/metabolic networks appear to have systematic differences in both the index of frustration and the transition to global order. these differences are interpretable in terms of the different functions of the various classes of networks. 