 Research
 Open Access
Priming nonlinear searches for pathway identification
 Siren R Veflingstad^{1, 2},
 Jonas Almeida^{3} and
 Eberhard O Voit^{3, 4}Email author
https://doi.org/10.1186/1742468218
© Veflingstad et al; licensee BioMed Central Ltd. 2004
 Received: 12 August 2004
 Accepted: 14 September 2004
 Published: 14 September 2004
Abstract
Background
Dense time series of metabolite concentrations or of the expression patterns of proteins may be available in the near future as a result of the rapid development of novel, highthroughput experimental techniques. Such time series implicitly contain valuable information about the connectivity and regulatory structure of the underlying metabolic or proteomic networks. The extraction of this information is a challenging task because it usually requires nonlinear estimation methods that involve iterative search algorithms. Priming these algorithms with highquality initial guesses can greatly accelerate the search process. In this article, we propose to obtain such guesses by preprocessing the temporal profile data and fitting them preliminarily by multivariate linear regression.
Results
The results of a smallscale analysis indicate that the regression coefficients reflect the connectivity of the network quite well. Using the mathematical modeling framework of Biochemical Systems Theory (BST), we also show that the regression coefficients may be translated into constraints on the parameter values of the nonlinear BST model, thereby reducing the parameter search space considerably.
Conclusion
The proposed method provides a good approach for obtaining a preliminary network structure from dense time series. This will be more valuable as the systems become larger, because preprocessing and effective priming can significantly limit the search space of parameters defining the network connectivity, thereby facilitating the nonlinear estimation task.
Keywords
 Piecewise Linear Model
 Piecewise Linear Regression
 Generalize Mass Action
 Biochemical System Theory
 Iterative Search Algorithm
Introduction
The rapid development of experimental tools like nuclear magnetic resonance (NMR), mass spectrometry (MS), tissue array analysis, phosphorylation of protein kinases, and fluorescence labeling combined with autoradiography on twodimensional gels promises unprecedented, powerful strategies for the identification of the structure of metabolic and proteomic networks. What is common to these techniques is that they allow simultaneous measurements of multiple metabolites or proteins. At present, these types of measurements are in their infancy and typically limited to snapshots of many metabolites at one time point (e.g., with MS; [1, 2]), to short time series covering a modest number of metabolites or proteins (e.g., with NMR [3, 4], 2d gels [5] or protein kinase phosphorylation [6]), or to tissue arrays [7] that permit the simultaneous highthroughput analysis of proteins in a single tissue section by means of antibody binding or MS. Nonetheless, it is merely a matter of time that these methods will be extended to relatively dense time series of many concentration or protein expression values. We will refer to these types of data as metabolic or proteomic profiles and to the time development of a single variable within such a composite profile as trace. The intriguing aspect of profiles is that they implicitly contain information about the dynamics and regulation of the pathway or network from which the data were obtained. The challenge for the mathematical modeler is thus to develop methods that extract this information and lead to insights about the underlying pathway or network.
In simple cases, the extraction of information can be accomplished to some degree by direct observation and interpretation of the shape of profiles. For instance, assuming a pulse perturbation from a stable steady state, Vance et al. [8] present guidelines for how relationships between the perturbed variable and the remaining variables may be deduced from characteristics of the resulting time profiles. These characteristics include the direction and timing of extreme values (i.e., the maximum deviation from steady state) as well as the slopes of the traces at the initial phase of the response. Torralba et al. [9] recently demonstrated that these guidelines, applied to a relatively small set of experiments, were sufficient to identify the first steps of an in vitro glycolytic system. Similarly, by studying a large number of perturbations, Samoilov et al. [10] showed that it is possible to quantify timelagged correlations between species and to use these to draw conclusions about the underlying network.
For larger and more complex systems, simple inspection of peaks and initial slopes is not feasible. Instead, the extraction of information from profiles requires two components. One is of a mathematical nature and consists of the need for a model structure that is believed to have the capability of capturing the dynamics of the underlying network structure with sufficient accuracy. The second is computational and consists of fitting this model to the observed data. Given these two components along with profile data, the inference of a network is in principle a regression problem, where the aim is minimization of the distance between the model and the data. If a linear model is deemed appropriate for the given data, this process is indeed trivial, because it simply requires multivariate linear regression, which is straightforward even in highdimensional cases. However, linear models are seldom valid as representations of biological data, and the alternative of a nonlinear model poses several taxing challenges.
First, in contrast to linear models, there are infinite possibilities for nonlinear model structures. In specific cases, the subject area from which the data were obtained may suggest particular models, such as a logistic function for bacterial growth, but in a generic sense there are hardly any guidelines that would help with model selection. One strategy for tackling this problem is the use of canonical forms, which are nonlinear structures that conceptually resemble the unalterable linear systems models, but are nonlinear. Canonical models have in common that they always have the same mathematical structure, no matter what the application area is. They also have a number of desirable features, which include the ability to capture a wide variety of behaviors, minimal requirements for a priori information, clearly defined relationships between network characteristics and parameters, and greatly enhanced facility for customized analysis.
The bestknown examples of nonlinear canonical forms are LotkaVolterra models (LV; [11]), their generalizations [12], and powerlaw representations within the modeling framework of Biochemical Systems Theory (BST; [13–15]), most notably Generalized Mass Action (GMA) systems and Ssystems. LotkaVolterra models have their origin in ecology and focus strictly on interactions between two species at a time. Wellstudied examples include competition processes between species, the dynamics of predators and prey, and the spread of endemic infections. In the present context it might seem reasonable to explore the feasibility of these models for the representation of the dynamics of proteins and transcription factor networks, but this has not been done so far.
The strict focus on twocomponent interactions in LV models has substantial mathematical advantages, but it has proven less convenient for the representation of metabolic pathways, where individual reaction steps depend on the substrate, but not necessarily on the product of the reaction, or are affected by more than two variables. A simple example of the latter is a bisubstrate reaction that also depends on enzyme activity, a cofactor and possibly on inhibition or modulation by some other metabolite in the system. These types of processes have been modeled very successfully with GMA and Ssystems. Between these two forms, the Ssystem representation has unique advantages for system identification from profiles, as was shown elsewhere [16–24] and will be discussed later in this article. In some sense, Karnaukhov and Karnaukhova [25] used a very simplified GMA system for biochemical system identification from dynamic data, in which all monosubstrate or bisubstrate reactions were of first order. This reduced the estimation to the optimization of rate constants, which the authors executed with an integral approach.
The inference of a nonlinear model structure from experimental data is in principle a straightforward "inverse problem" that should be solvable with a regression method that minimizes the residual error between model and data. In practice, however, this process is everything but trivial (cf. [26]) as it almost always requires an iterative search algorithm with all its numerical challenges, such as the existence of multiple local minima and failure to converge. Recent attempts of ameliorating this problem have included Bayesian inference methods [27], similarity measures and correlation [28], mutual information [29], and genetic algorithms [30]. An indication of the complexity of nonlinear estimation tasks and their solutions is a recent pathway identification involving an Ssystem with five variables, which was based on a genetic algorithm [21]. The algorithm successfully estimated the parameter values, but although the system under study was relatively small and noise free, each loop in the algorithm took 10 hours on a cluster of 1,040 Pentium III processors (933 MHz). It is quite obvious that such an approach cannot be scaled up to systems of dozens or hundreds of variables.
Nonlinear estimation methods have been studied for a long time, and while computational and algorithmic efficiency will continue to increase, the combinatorial explosion of the number of parameters in systems with increasingly more variables mandates that identification tasks be made easier if larger systems are to be identified. One important possibility, which we pursue here, is to prime the iterative search with highquality starting conditions that are better than naïve defaults. Clearly, if it is possible to identify parameter guesses that are relatively close to the true, yet unknown solution, the algorithm is less likely to get trapped in suboptimal local minima. We are proposing here to obtain such initial guesses by preprocessing the temporal profile data and fitting them preliminarily by straightforward multivariate linear regression. The underlying assumption is that the structural and regulatory connectivity of the network will be reflected, at least qualitatively, in the regression coefficients. D'haeseleer et al. [31] explored a similar approach for analyzing mRNA expression profiles, but could not validate their results because they lacked a mechanistic model of gene expression. Furthermore, because of the unique relationship between network structure and parameters in Ssystem models (see below), we will demonstrate that it is possible to translate the regression coefficients into constraints on the parameter values of an Ssystem model and thereby to reduce the parameter search space very dramatically.
Several other groups have recently begun to target network identification tasks with rather diverse strategies. Chevalier et al. [32] and DiazSierra and coworkers [33, 34] proposed an identification approach that is similar to the one proposed here in some aspects, though not in others. These authors also used linearization of a nonlinear model, but based their estimation on measured time developments of the system immediately in response to a small perturbation. These measurements were used to estimate the Jacobian of the system at the steady state. In contrast to this focus on a single point, we are here using smoothed longterm time profiles and do not necessarily require system operation at a steady state. Also using linearization, Gardner et al. [35] recently proposed a method of network identification by multiple regression. However, they only considered steadystate measurements as opposed to temporal profiles. It is known from theoretical analyses (e.g., [15, 36]) that different dynamical models may have the same steady state and that therefore steadystate information alone is not sufficient for the full characterization of a network. Mendes and Kell [37] used a neural network approach for an inverse problem in metabolic analysis, but their target system was very small and fully known in structure. Furthermore, their data consisted of a "large number of steadystate simulations", rather than the limited number of time traces on which our analysis is based. Chen et al. [38] used neural networks and cubic splines for smoothing data and identifying rate functions in otherwise linear massbalance models.
Methods
The behavior of a biochemical network with n species can often be represented by a system of nonlinear differential equations of the generic form
where X is a vector of variables X_{ i }, i = 1, ..., n, f is a vector of nonlinear functions f_{ i }, and μ is a set of parameters. If the mathematical structure of the functions f_{ i }is known, the identification of the network consists of the numerical estimation of μ. In addition to the challenges associated with nonlinear searches mentioned above, this estimation requires numerical integration of the differential equations in (1) at every step of the search. This is a costly process, requiring in excess of 95% of the total search time; if the differential equations are stiff, this percentage approaches 100% [39]. A simplification, which circumvents the problem of integration, consists of substituting the system of differential equations with decoupled algebraic equations by replacing the differentials on the lefthand side of Eq. (1) with estimated slopes [16, 17]. Thus, if the system consists of n differential equations, and if measurements are available at N time points, the decoupling leads to n × N algebraic equations of the form
It may be surprising at first that it is valid to decouple the tightly coupled system of nonlinear differential equations. Indeed, this is only justified for the purpose of parameter estimation, where the decoupled algebraic equations simply provide numerical values of variables (metabolites or proteins) and slopes at a finite set of discrete time points. The experimental measurements thus serve as the "data points," while the parameters μ_{ ij }are the "unknowns" that need to be identified.
The quality of this decoupling approach is largely dependent on an efficient and accurate estimation of slopes from the data. Since the data must be expected to contain noise, this estimation is a priori not trivial. However, we have recently shown [23, 39] that excellent estimates can be obtained by smoothing the data with an artificial neural network and computing the slopes from the smoothed traces (see Appendix for detail).
Different Linearization Approaches
The smoothing and decoupling approach reduces the cost of finding a numerical solution of the estimation task considerably. Nonetheless, algorithmic issues associated with local minima and the lack of convergence persist and can only be ameliorated with good initial guesses. To this end, we linearize the model f in Eq. (1) about one or several reference states. As long as the system stays close to the given reference state(s), this linearization is a suitable and valid approximation. We consider four options: (I) linearization of absolute deviations from steady state; (II) linearization of relative deviations from steady state; (III) piecewise linearization; and (IV) LotkaVolterra linearization.
Option (I) is based on deviations of the type z_{ i }= X_{ i } X_{ ir }, where X_{ ir }denotes the value at a reference state of choice. If the reference state is chosen at a stable steady state, the firstorder Taylorapproximation is given by
where A is the n × n Jacobian with elements a_{ ij }= (df_{ i }/ dX_{ j }) calculated at X_{ r }(cf. [32–34]). If the reference state is not chosen at a steady state, the equation contains an additional constant term a_{i 0}, which is equal to f_{ i }(X_{ r }).
For option II, we define a new variable u_{ i }= z_{ i }/X_{ ir }. At a steady state, this yields the linear system
where A' is an n × n matrix in which a'_{ ij }= (X_{ jr }/ X_{ ir })·a_{ ij }.
A general concern regarding linearization procedures is the range of validity of sufficiently accurate representation, which is impossible to define generically. From an experimental point of view, the perturbations from steady state must be large enough to yield measurable responses. This may require that they be at the order of 10% or more. Depending on the nonlinearities in f, a perturbation of this magnitude may already lead to appreciable approximation errors. While this is a valid argument, it must be kept in mind that the purpose of this priming step is simply to detect the topological structure of connectivity and not necessarily to estimate precise values of interaction parameters. Simulations (see below) seem to indicate that this detection is indeed feasible in many cases, even if the deviations are relatively large.
In order to overcome the limitation of small perturbations, a piecewise linear regression (option III) may be a superior alternative. In this case, we subdivide the dataset into appropriate time intervals and linearize the system around a chosen state within each subset. Most (or all) reference states are now different from the steady state, with the consequence that Eq. (3) has a constant term a_{i 0}, which is equal to f_{ i }(X_{ r }). The choice of subsets and operating points offers further options. In the analysis below, we use the locations of extreme values (maximum deviation from steady state) of the variables as the breakpoints between different subsets. Thus, a variable with a maximum and a later minimum has its time course divided into three subsets.
The fourth alternative (option IV) is a LotkaVolterra linearization. In a LotkaVolterra model, the interaction between two species X_{ i }and X_{ j }is assumed to be proportional to the product X_{ i }X_{ j }[11]. Furthermore accounting for linear dependence on the variable of interest itself, the typical LotkaVolterra equation for the rate of change in X_{ i }is
The righthand side of this nonlinear differential equation becomes linear if both sides are divided by X_{ i }, which is usually valid in biochemical and proteomic systems, because all quantities of interest are nonzero. Thus, the differentials are again replaced by estimated slopes, the slopes are divided by the corresponding variable at each time point, and fitting the nonlinear LV model to the time profiles becomes a matter of linear regression that does not even require the choice of a reference state. The quality of this procedure is thus solely dependent on the quality of the data and ability of the LV model to capture the dynamics of the observed network. It is known (e.g., [11, 40]) that the mathematical structure of LV models is rich enough to model any nonlinearities, if sufficiently many equations are included. However, there is no general information about the quality of fit in particular modeling situations.
Regression
Transformation of data for regression analysis
RESPONSE VARIABLE  PREDICTOR VARIABLE  

A. Absolute deviation from a reference state  y_{ i }=  x_{ i }= X_{ i } X_{ ir } 
B. Relative deviation from a reference state 


C. LotkaVolterra system 
 x_{ i }= X_{ i } 
The result of the regression is a matrix of coefficients that indicate to what degree a metabolite X_{ j }affects the dynamics (slope) of another metabolite X_{ i }. In particular, a coefficient that is zero or close to zero signals that there is no significant effect of X_{ j }on the slope of X_{ i }. By the same token, a coefficient that is significantly different from zero suggests the presence of an effect, and its value tends to reflect the strength and direction of the interaction. In either case, the coefficients computed from the linear regression provide valuable insight into the connectivity of the network. Furthermore, the estimated coefficients provide constraints on the parameter values of the desired nonlinear model f. Indeed, if f consists of an Ssystem model, the coefficients estimated from the regression can be converted into combinations of Ssystem parameters, as is demonstrated in the following theoretical section and illustrated later with a specific example.
Relationships between Estimated Regression Coefficients and Ssystem Parameters
The regression analysis yields coefficients that offer information on the connectivity of the network of interest. It also provides clues about the parameter values of the underlying nonlinear network model f in Eq. (1) if this model has the form of an Ssystem. To determine the relationships between the regression coefficients and the parameters of the Ssystem, it is convenient to work backwards by computing the different types of linearizations discussed before for the particular case of Ssystem models. This derivation is simply a matter of applying Taylor's theorem.
In the Ssystem formalism, the rate of change in each pool (variable) is represented as the difference between influx into the pool and efflux out of the pool. Each term is approximated by a product of powerlaw functions, so that the generic form of any Ssystem model is
where n is the number of state variables [13, 14]. The exponents g_{ ij }and h_{ ij }are called kinetic orders and describe the quantitative effect of X_{ j }on the production or degradation of X_{ i }, respectively. A kinetic order of zero implies that the corresponding variable X_{ j }does not have an effect on X_{ i }. If the kinetic order is positive, the effect is activating or augmenting, and if it is negative, the effect is inhibiting. The multipliers α _{ i }and β _{ i }are rate constants that quantify the turnover rate of the production or degradation, respectively.
If the Taylor linearization is performed at a steady state, the production term of the Ssystem model equals the degradation term. The absolute deviation of the first option, z_{ i }= X_{ i } X_{ is }, where the subscript s denotes the value of the variable at steady state, then leads directly to
where
c_{ ij }= g_{ ij } h_{ ij },
Comparing the expression in Eq. (6) with the linear regression results, one sees immediately that each coefficient a_{ ij }in Eq. (3) corresponds to the product of F_{ ij }and c_{ ij }:
a_{ ij }= F_{ ij }c_{ ij }. (7)
Thus, once the regression has been performed and the coefficients a_{ ij }have been estimated, the parameters of the corresponding Ssystem are constrained – though not fully determined – by Eq. (7). In particular, Eq. (7) does not allow a distinction between various combinations of g_{ ij }and h_{ ij }, as long as the two have the same difference. For instance, reinterpreting the regression coefficients as Ssystem parameters does not differentiate between the overall absence of effect of X_{ j }on X_{ i }(g_{ ij }= h_{ ij }= 0) and the same effect of X_{ j }on both the production and degradation of X_{ i }(g_{ ij }= h_{ ij }≠ 0). This observation is related to the observation of Sorribas and Cascante [36] that steadystate measurements are insufficient for completely identifying an Ssystem model.
Relative deviations from steady state, u_{ i }= (X_{ i } X_{ is }) / X_{ is }, in option II, are assessed in an analogous fashion. In this case one obtains
where
c_{ ij }= g_{ ij } h_{ ij },
[41]. Again, the Ffactors F_{ i }are positive, while c_{ ij }may be either positive or negative.
The piecewise linear model for an Ssystem is easily derived as well. It is given as
where X_{ jr }denotes the value of the variable at the reference state. This case also includes the situation of a single approximation, which however is not necessarily based on a steadystate operating point.
In the case of the LotkaVolterra linearization, the correspondence between computed regression coefficients and Ssystem parameters is determined most easily by dividing the Ssystem equations by the corresponding X_{ i }and then linearizing around an operating point. The resulting expressions become especially simple if this point is chosen as the steady state. In this case, the relationship between the parameters of the LV system and the Ssystem are
where c_{ ij }= g_{ ij } h_{ ij }.
Results
We applied the methods described in the previous sections to simulated time profiles obtained from the small gene network in Figure 1a. Hlavacek and Savageau [42] modeled this network as an Ssystem with five differential equations (Figure 1b), and Kikuchi et al. [21] used it recently for exploring computational features of their proposed structure identification algorithm. The benefit of working with a known model is that complete information is available about both its structure and parameter values. In particular, it is possible to perform any number of experiments and to produce data and slopes with predetermined noise levels, which is not typically possible with real data. For this analysis, we thus used simulated noise free "data," which allowed us to skip the neural network step of smoothing [23, 39].
To generate time profiles, the system was implemented with the parameter values published by Hlavacek and Savageau [42], and as in the analysis of Kikuchi et al. [21], the model was initialized with various perturbations from steady state and numerically integrated over a sufficient time horizon to allow the system to return to the steady state.
Preliminary Analysis
Maximal information about the network is obtained when every variable is perturbed sequentially. Experimentally, such perturbations could be implemented with modern methods of RNA interference [43] or, for biotechnological purposes, in a chemostat [9]. In our model case, we can actually identify all kinetic orders that are zero in the original model, and this amounts to determining the connectivity of the pathway. The only relationship this analysis does not pick up is the effect of X_{2} on X_{3}. This result is not surprising, because the effect of X_{2} is the same on both the production and degradation of X_{3}, which leads to cancellation. It is noted that this analysis does not necessarily distinguish between transfer of mass and a positive modulation, because both result in a positive effect on a variable. In a realistic situation, biological knowledge may exclude one of the two options, as in this case, where modulation is the only possibility for the effect of X_{3} on both X_{1} and X_{4}, because the former is a protein and the latter are RNA transcripts. For the mathematical model in the Ssystem form, this is not an issue, as both types of influence are included in the equations in the same way (as a positive kinetic order).
Regression Analysis
While Vance's method works well in this simple noisefree system, it is not scalable to larger and more complex systems. The next step of our analysis is therefore regression according to the four options presented above and with a number of simulated datasets of the gene network that differ in the variable to be perturbed and the size of the perturbation. Because the illustration here uses a known model and artificial data, it is easy to compute the true regression coefficients through differentiation of the Ssystem model. These coefficients can be used as a reference for comparisons with coefficients computed from the entire time traces, which mimics the estimation process for (smoothed) actual data.
Options I, II and IV
The results for three of the options (I, II and IV) can be summarized in the following three points, while the piecewise linear model will be discussed afterwards.
Comparison of computed and estimated coefficients
Computed coefficients  Estimated coefficients  

a10  0  0.0000 
a11  14.6780  14.3647 
a12  0  0.1466 
a13  7.3390  7.3414 
a14  0  0.2165 
a15  7.3390  7.1723 
a20  0  0.0000 
a21  14.6780  14.6119 
a22  14.6780  14.6540 
a23  0  0.0009 
a24  0  0.0494 
a25  0  0.0309 
a30  0  0.0000 
a31  0  2.3527 
a32  0  1.3989 
a33  27.2517  27.9204 
a34  0  1.7491 
a35  0  0.9955 
a40  0  0.0000 
a41  0  2.0843 
a42  0  1.0925 
a43  18.5664  19.0295 
a44  18.5664  20.2112 
a45  9.2832  8.3594 
a50  0  0.0000 
a51  0  0.4026 
a52  0  0.1384 
a53  0  0.0059 
a54  18.5664  18.8987 
a55  18.5664  18.7852 
Comparison of the different linearization options (I, II and IV)
I. Absolutedeviation  II. Relative deviation  IV. LotkaVolterra  

a10  0.0000  0.0000  14.4748 
a11  14.3647  14.3647  18.9581 
a12  0.1466  0.1466  0.6836 
a13  5.3878  7.3414  7.3367 
a14  0.1712  0.2165  0.4694 
a15  5.6702  7.1723  7.4981 
a20  0.0000  0.0000  0.0144 
a21  14.6119  14.6119  19.8910 
a22  14.6540  14.6540  19.9277 
a23  0.0006  0.0009  0.0001 
a24  0.0390  0.0494  0.0472 
a25  0.0245  0.0309  0.0335 
a30  0.0000  0.0000  26.4020 
a31  3.2058  2.3527  2.8725 
a32  1.9062  1.3989  1.7989 
a33  27.9204  27.9204  26.6164 
a34  1.8842  1.7491  1.5871 
a35  1.0724  0.9955  0.9692 
a40  0.0000  0.0000  8.0270 
a41  2.6365  2.0843  6.3364 
a42  1.3820  1.0925  4.1579 
a43  17.6654  19.0295  19.0005 
a44  20.2112  20.2112  23.1319 
a45  8.3594  8.3594  7.7047 
a50  0.0000  0.0000  0.0869 
a51  0.5092  0.4026  0.6617 
a52  0.1751  0.1384  0.4441 
a53  0.0055  0.0059  0.0003 
a54  18.8987  18.8987  20.2939 
a55  18.7852  18.7852  20.2152 
The effect of the size of the perturbation
Computed  5 %  10 %  50 %  200 %  

a10  0  0.0000  0.0000  0.0001  0.0008 
a11  14.6780  14.3647  14.1817  13.1496  11.3439 
a12  0  0.1466  0.1429  0.0671  0.5735 
a13  7.3390  7.3414  7.3438  7.3598  7.3735 
a14  0  0.2165  0.3673  1.2462  2.7619 
a15  7.3390  7.1723  7.0780  6.4846  5.2501 
a20  0  0.0000  0.0000  0.0000  0.0003 
a21  14.6780  14.6119  14.5748  14.4207  14.5029 
a22  14.6780  14.6540  14.6623  14.7503  15.1862 
a23  0  0.0009  0.0016  0.0054  0.0070 
a24  0  0.0494  0.0839  0.2494  0.3462 
a25  0  0.0309  0.0464  0.1119  0.0951 
a30  0  0.0000  0.0000  0.0004  0.0038 
a31  0  2.3527  4.5412  18.2307  46.8953 
a32  0  1.3989  2.6336  9.8422  24.4004 
a33  27.2517  27.9204  28.5955  34.0204  54.4047 
a34  0  1.7491  3.4009  14.0961  39.3252 
a35  0  0.9955  1.8949  7.0627  15.4759 
a40  0  0.0000  0.0000  0.0001  0.0001 
a41  0  2.0843  3.7814  14.7316  41.5863 
a42  0  1.0925  1.7693  5.5766  13.2688 
a43  18.5664  19.0295  19.4964  23.2397  37.1866 
a44  18.5664  20.2112  21.6608  31.4631  58.1065 
a45  9.2832  8.3594  7.6404  3.2226  6.5808 
a50  0  0.0000  0.0000  0.0001  0.0015 
a51  0  0.4026  0.6581  2.5848  10.1097 
a52  0  0.1384  0.0830  0.1317  0.1582 
a53  0  0.0059  0.0110  0.0435  0.0879 
a54  18.5664  18.8987  19.1602  21.0620  27.2722 
a55  18.5664  18.7852  18.9201  20.0013  24.0836 
Option III
Results for piecewise linear regression
Interval 1  Interval 2  Interval 3  

a10  0.1315  0.0419  0.0000 
a11  42.3980  14.1738  14.5490 
a12  0.0000  0.8010  0.0464 
a13  8.9105  7.3653  7.6299 
a14  12.7757  0.3340  0.1386 
a15  3.3476  6.9121  7.2940 
a20  0.0567  0.0197  0.0000 
a21  1.1939  14.4913  14.6792 
a22  32.3300  14.5116  14.6784 
a23  0.6133  0.0057  0.0205 
a24  7.0917  0.1016  0.0018 
a25  7.9313  0.1047  0.0067 
a30  0.7858  0.0181  0.0000 
a31  130.3724  0.2358  0.0021 
a32  0.0000  0.3616  0.0007 
a33  20.7724  27.6129  27.2551 
a34  62.1525  0.3496  0.0027 
a35  19.1470  0.1984  0.0006 
a40  0.3164  0.0709  0.0000 
a41  13.6819  1.1412  0.0115 
a42  0.0000  2.1478  0.0015 
a43  19.8295  18.8534  18.6927 
a44  13.3654  19.5811  18.5494 
a45  7.2135  8.0985  9.2792 
a50  0.1617  0.0393  0.0000 
a51  149.5199  0.8195  0.0250 
a52  160.3341  0.8175  0.0074 
a53  5.7537  0.0580  0.0304 
a54  85.3050  19.0394  18.5356 
a55  53.9745  19.1183  18.5623 
Degree of Similarity as a Measure of Reliability
Collective inference of the gene network based on results from all linearizations
X 1  X 2  X 3  X 4  X 5  

X1   (100 %)  0 (67 %)  + (100 %)  0 (83 %)   (100 %) 
X2  + (100 %)   (100 %)  0 (100 %)  0 (83 %)  0 (83 %) 
X3  ?  ?   (100 %)  ?  ? 
X4  + (67 %)   (67 %)  + (100 %)   (100 %)   (100%) 
X5   (83 %)  0 (83 %)  0 (83 %)  + (100 %)   (100 %) 
Constraining the Parameter Values
In addition to reflecting the connectivity, the coefficients provide likely parameter ranges or likely constraints on parameter values of the true model. As an example, consider variable X_{1}. Table 6 indicates that the variables having a significant effect are X_{1}, X_{3} and X_{5}. If so, the linear model in Eq. (8) suggests the following:
Discussion
Identifying the structure of metabolic or proteomic networks from time series is a task that most likely will require large, parallelized computational effort. The search space for the algorithms is typically of high dimension and unknown structure and very often contains numerous local minima. This generic and frequent problem may be ameliorated if the search algorithm is provided with good initial guesses and/or constraints on admissible parameter values. Here, we have shown that linear regression may provide such information directly from the types of data to be expected from future experiments. For illustrative purposes, we used artificial data from a known network, but all methods are directly applicable to actual profile data and scaleable to large systems.
The coefficients estimated from the different regressions reflect the effect of one variable on another surprisingly well and thus provide a simple fashion of prescreening the connectivity of the network. In addition, the estimated coefficients provide constraints on the parameter values, if the alleged nonlinear model has the form of an Ssystem. To explore the preassessment of data as fully as feasible, we studied four linearization strategies: using an absolute deviation from steady state; a relative deviation from steady state; piecewise linearization; and LotkaVolterra linearization. Interestingly, all models gave qualitatively similar results for the analyzed example, and this degree of similarity may provide a measure of how reliable the identified connections are. Specifically, of the 25 possible connections in the small gene network studied, 19 were identified correctly in at least 83 % of the regression analyses.
A concern of any linearization approach is the validity of the linear approximation. However, as long as the perturbation from steady state remains relatively small, the estimated linear model is likely to be a good fit of the actual nonlinear model, at least qualitatively. This limitation may furthermore be alleviated by fitting the profile data in a piecewise linear fashion. As most reference states in this case are different from the steady state, this strategy has the added benefit that more of the true relationships within the nonlinear model are likely to be preserved. As an alternative, one could explore the performance of the socalled "loglinear" model, which is linear in logtransformed variables [44].
The LotkaVolterra linearization did not perform as well as expected with regard to large perturbations. This may be a consequence of the particular example, which was originally in Ssystem form rather than in a form more conducive to the LV structure, which emphasizes interactions between pairs of variables. Since it is easy to perform the LV analysis along with the other regressions discussed here, it may be advisable to execute all four analyses.
The illustrative model used for testing the procedure consisted of a relatively small system with only five variables and relatively few interactions. Nonetheless, one should recall that this very system required substantial identification time in a direct estimation approach [21]. In order to check how scaleable the results of the proposed linearization method are, the method should be tested on larger systems. Some preliminary analyses suggest that the method works well, but that the likelihood of misidentified connections may grow with the size of the system, as one might expect. At the same time, experience with actual biological networks, for instance in ecology and metabolism, suggests that larger systems are often more robust in a sense that they do not deviate as much from the steady state as smaller systems. If this trend holds in general, the linearization becomes a more accurate representation as larger networks are being investigated and the proposed methods will therefore yield more reliable initial indicators of network connectivity. Independent of these issues, the methods proposed here will very likely be more valuable for bigger systems than other methods that are presently available, because without some preprocessing of the data and effectively priming the search, as it is proposed here, the combinatorial explosion will most certainly gain the upper hand eventually.
Appendix
It was recently shown that good parameter estimates of Ssystem models from metabolic profiles might be obtained by training an artificial neural network (ANN) directly with the experimental data. The result of this training is a socalled universal function which smoothes the data with predetermined precision and also allows the straightforward computation of slopes that can be used for network identification purposes. This appendix briefly outlines the procedure; details can be found in Almeida [45] and Voit and Almeida [24]. The ANN consists of three layers; one input layer, one hidden layer and one output layer. The input layer consists of the measurement time points, the hidden layer has no direct biological interpretation, and the output layer contains the metabolite concentrations or levels of protein expression that the ANN is being trained to represent. The node values of the ANN in the hidden layer are calculated from a linear combination of input values with different weights according to a multivariate logistic equation. Similarly, the values of the output layer are determined from linear combinations of the hidden node values with different weights, again using a multivariate logistic function. It is known that this type of nested multivariate logistic function has unlimited flexibility in modeling nonlinearities [46].
Noise and sample size do not have a devastating effect on the results of the ANNmethod, as long as the true trend is well represented [39]. In fact, the ANN approach provides an unlimited number of sampling points, as values at any desired time points may be estimated from the universal output function. Finally, the calculation of the slopes of the smooth output functions is mathematically unwieldy, but computationally straightforward.
The use of the entire time course is in stark contrast to earlier methods of parameter estimation and structure identification in metabolic networks. Mendes and Kell [37] applied their ANNbased parameter estimation to steadystate data, while we are using time profiles.
Chevalier and coworkers [32] first fitted the nonlinear solution with a linear model (as shown in Eq. 3), expressed this solution in terms of eigenvectors and eigenvalues, and then obtained the slopes by numerical differentiation. Sorribas et al. [47] suggested a variation on this approach, based on discretizing the solution of Eq. (3) as
z(t_{k + 1}) = z(t_{ k })exp(h·A), (A1)
where h is the step size. The problem is thereby reduced to a mulitilinear regression in which the matrix Φ = exp(h·A) is the output. Instead of estimating the slopes, they obtain the Jacobian directly by expanded in its Taylorseries. This approach yields a faster convergence to the elements of the Jacobian than the one suggested by Chevalier et al. [32], but the regression of Eq. (A1) is very sensitive to noise and missing data points.
Our approach takes advantage of the entire time course and is therefore less sensitive to the particularities of assessing a system at a single point. The ANN itself does not provide much insight, because it is strictly a blackbox model, but it is a valuable tool for controlling problems that are germane to any data analysis, namely noise, measurement inaccuracies, and missing data.
Declarations
Acknowledgments
This research was carried out during S.R.V.'s scientific visit at the Medical University of South Carolina. The work was supported by a Quantitative Systems Biotechnology grant (BES0120288; E.O. Voit, PI) from the National Science Foundation, a National Heart, Lung and Blood Institute Proteomics Initiative through contract N01HV28181 (D. Knapp, PI), and an Interdisciplinary USC/MUSC grant (E.P. Gatzke, PI). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the sponsoring institutions.
Authors’ Affiliations
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