 Method
 Open access
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Flux balance analysis accounting for metabolite dilution
Genome Biology volumeÂ 11, ArticleÂ number:Â R43 (2010)
Abstract
Flux balance analysis is a common method for predicting steadystate flux distributions within metabolic networks, accounting for the growth demand for the synthesis of a predefined set of essential biomass precursors. Ignoring the growth demand for the synthesis of intermediate metabolites required for balancing their dilution leads flux balance analysis to false predictions in some cases. Here, we present metabolite dilution flux balance analysis, which addresses this problem, resulting in improved metabolic phenotype predictions.
Background
A practical approach to gaining biological understanding of complex metabolic networks requires the development of mathematical modeling, simulation, and analysis techniques. Traditional modeling techniques are based on mathematical approaches that require detailed and accurate information regarding reaction kinetics as well as enzyme and metabolite concentrations [1, 2]. The lack of sufficient data limits the current applicability of such methods to smallscale systems. This hurdle is surpassed through the use of constraintbased modeling (CBM), which serves to analyze the functionality of genomescale metabolic networks by relying solely on simple physicalchemical constraints [3, 4]. Genomescale CBM models have already been constructed for more than 50 organisms [5], including common model microorganisms [6, 7], industrially relevant microbes [8â€“11], various pathogens [12â€“15], and recently for human cellular metabolism [16]. Flux balance analysis (FBA) is a key computational approach within the CBM modeling framework [17â€“19] and is frequently used to successfully predict various phenotypes of microorganisms, such as their growth rates, uptake rates, byproduct secretion, and knockout lethality (see [3, 5, 20] for reviews).
Traditional kinetic models of cellular metabolism are formulated as a set of differential equations that compute the time derivative of metabolite concentrations (denoted by ) as dependent on reaction rates (denoted by ; which, in turn, depend on metabolic concentration and kinetic constants, denoted by ) and metabolite dilution due to cellular growth (with Î¼ denoting the growth rate) [21]:
where S is an m Ã— n stoichiometric matrix, m is the number of metabolites, n is the number of reactions, and S_{ ij }represents the stoichiometric coefficient of metabolite i in reaction j. A precise solution to Equation 1 requires determination of the kinetic parameters , which are generally unavailable, resulting in the development of the alternative CBM approach. In CBM, an entire space of possible solutions for the flux distribution
is postulated, considering that the metabolic system is constrained by physicochemical, environmental and regulatory constraints. In FBA, this solution space is constrained by the assumption of a quasi steadystate, under which stoichiometric massbalance constraints enforce constant concentrations of intermediate metabolites over time:
The uptake and secretion of a predefined set of metabolites from and to the environment is facilitated via the definition of exchange reactions in the stoichiometric matrix S [3]. A pseudo growth reaction is defined to simulate the utilization of metabolites during growth, consuming the most abundant biomass constituents based on experimentally determined concentrations (that is, the jth component in denotes the steadystate concentration of metabolite j). The objective of FBA is to find a steadystate flux distribution, , satisfying Equation 2 alongside additional enzymatic directionality and capacity constraints [3], together permitting a maximal growth rate Î¼. Accounting only for linear constraints, the resulting space of feasible flux distribution described by FBA is convex (forming a highdimensional polytope), in which optimal biomass producing solutions can be efficiently searched for via linear programming (LP).
The employment of a pseudo growth reaction in FBA to represent the utilization of metabolites as part of growth poses two fundamental problems. First, the metabolite composition of cellular biomass significantly varies across different growth media, genetic backgrounds and growth rates [22â€“24]. Indeed, previous work by Pramanik and Keasling [22, 23] has shown that using the correct experimentally measured biomass composition of Escherichia coli under different growth media and growth rates significantly improves FBA flux predictions. However, as FBA is commonly applied to probe metabolic behavior under diverse genetic and environmental conditions for which no metabolite concentration data are available, it has become common practice to employ a constant biomass composition across all conditions [25]. Second, the growth reaction in various CBM models commonly accounts for no more than a few dozen metabolites, for which measured concentrations are available under a specific condition [23]. Ignoring the growthassociated dilution of the remaining metabolites (those not included in the biomass composition in use; required by Equation 1) may result in the prediction of biologically implausible flux distributions, leading to false predictions of gene essentiality and growth rates, as shown in the Results. This problem has been recently addressed by Kruse and EbenhÃ¶h [26], who suggested a method that is based on network expansion to compute the set of producible metabolites under a given growth medium. This method, however, does not enable the prediction of feasible flux distributions that account for the growthassociated dilution of all intermediate metabolites. Another approach, recently suggested by Martelli et al. [27], predicts metabolic fluxes based on Von Neumann's model, which maximizes the growth rate in a metabolic network without assuming massbalance nor utilizing prior knowledge of a biomass composition. However, similarly to FBA, flux distributions predicted by this method do not fully account for the growthassociated dilution of all intermediate metabolites.
In this paper we describe a variant of FBA, metabolite dilution flux balance analysis (MDFBA), which aims to predict metabolic flux distributions by accounting for the dilution of all intermediate metabolites that are synthesized under a given condition. As shown below, accounting for growth dilution of intermediate metabolites is especially important for metabolites that participate in catalytic cycles, many of them being metabolic cofactors. Since CBM assumes a steadystate flux distribution and does not predict the actual concentration of the intermediate metabolites, we consider a uniform minimal dilution rate for all intermediate metabolites produced via a nonzero flux through some reaction (assuming a uniform concentration for all intermediate metabolites, following [28]).
Figure 1 demonstrates an example network for which FBA and MDFBA predict different flux distributions, leading to different growth rate and gene essentiality predictions. The biomass in this network is metabolite B, while the input metabolites available in the growth medium are A and X in Figure 1a, and only A in Figure 1b. The synthesis of the biomass precursor B is facilitated via two alternative pathways: through an efficient pathway via v_{4}, producing one molecule of B per molecule of A; or through an inefficient pathway via reactions v_{2} and v_{3}, producing one molecule of B per two molecules of A. Reaction v_{4} requires the presence of a cofactor metabolite C, which is recycled via reaction v_{8} and synthesized via reactions v_{6} and v_{7}. Thus, in MDFBA, the activation of the efficient pathway for synthesizing B via v_{4} enforces de novo synthesis of cofactor C via v_{6} and v_{7} to balance the dilution of this cofactor (Figure 1a, red solid arrows). By contrast, since FBA does not account for metabolite dilution, it would predict a biologically implausible flux distribution in which the steadystate concentration of the cofactor C is maintained via reaction v_{8}, without predicting the growthassociated demand for de novo synthesis of this cofactor (Figure 1a, b, blue dotdash arrows). The different flux distributions predicted by FBA and by MDFBA under the two growth media yield different growth rate and enzyme essentiality predictions. FBA predicts the activation of the efficient biosynthetic pathway for synthesizing metabolite B under both growth media, resulting in the same growth rate prediction under the two media. MDFBA, on the other hand, predicts the activation of the efficient biosynthetic pathway when metabolite X is present in the growth medium (with a growth rate prediction similar to that of FBA; Figure 1a) and the activation of the inefficient pathway when metabolite X is absent (resulting in a lower growth rate; Figure 1b). When metabolite X is present in the growth medium, MDFBA, unlike FBA, predicts that the biosynthetic pathway for the production of cofactor C is activated, with the reactions v_{6} and v_{7} being essential for achieving maximal growth rate (Figure 1a). When X is absent from the growth medium, FBA predicts the activity of the efficient pathway through v_{4} and v_{8}, with the corresponding enzymes essential for obtaining a maximal growth rate. MDFBA, however, predicts the inactivation of this efficient pathway and hence the inessentiality of v_{4} and v_{8}, while predicting the enzymes in the less efficient pathway v_{2} and v_{3} to be essential for growth (Figure 1b).
Next, we describe the implementation of MDFBA as a mixedinteger linear programming (MILP) optimization problem and demonstrate its applicability in predicting metabolic phenotypes, outperforming the commonly used FBA method.
Results
MDFBA  accounting for growthassociated dilution of all intermediate metabolites
Our method, MDFBA, aims to predict a feasible flux distribution through a metabolic network under a given environmental and genetic condition, by maximizing the production rate of the biomass (that is, the flux through the biomass reaction) while satisfying a stoichiometric massbalance constraint, accounting for the growthassociated dilution of all produced intermediate metabolites, and satisfying enzymatic directionality and capacity constraints embedded in the model (similarly to FBA). MDFBA is formulated as a MILP problem as defined in the Materials and methods.
Applying MDFBA to predict metabolic phenotypes in Escherichia coli
As a benchmark for the prediction performance of MDFBA, we applied it to the genomescale metabolic network model of E. coli [6] to predict growth rates and gene essentiality under a diverse set of growth media and gene knockouts. The model of Feist et al. [6] accounts for 1,260 metabolic genes, 2,382 reactions and 1,668 metabolites.
As an initial validation, we applied both MDFBA and FBA to predict E. coli's growth rate for 91 gene knockout strains under 125 different media, yielding a total of 11,375 growth conditions for which measured optical density (OD) data are available via a phenotypic array in the ASAP database [29]. Each medium included a fixed set of metabolites (oxygen, phosphate, water, sulfate, carbon dioxide, hydrogen and metal ions) and alternating carbon and nitrogen sources (the full list of growth conditions (media and gene knockouts) as well as the experimental OD values are available in Additional files 1 and 2, respectively). Different gene knockouts were modeled by forcing a zero flux through the corresponding enzymecatalyzed reactions [28]; different growth media were modeled by changing the bounds on metabolite uptake from the environment based on specification of the available metabolic nutrients in each medium [28]. Both FBA and MDFBA predicted no growth for the wildtype strain under 13 growth media and hence these media were removed from further analysis. In an additional 16 growth media the correlation between the growth rates predicted by FBA and MDFBA across all knockout strains was significantly high (Spearman r > 0.7) and hence these media were also removed from further comparison of the two methods (the results presented below are insensitive to specific choice of a Spearman correlation threshold). For each deletion strain, a Spearman correlation was calculated between the predicted growth rates and the measured OD values across the remaining 96 different growth media. For 10 of the 91 gene deletion strains, both FBA and MDFBA falsely predicted zero growth across all media and these strains were removed from further analysis. The median Spearman correlation obtained by MDFBA was found to be slightly higher than that obtained via FBA (Wilcoxon test Pvalue = 0.0145; Figure 2). Several limitations of the MDFBA method currently restrict its ability to markedly improve the growth rate predictions, as discussed below. Still, in some interesting specific cases MDFBA outperforms FBA; for example, we examined two minimal growth media, NacetylDmannosamine and NacetylDglucosamine, under which the latter yields a higher measured growth rate across all knockout strains, while FBA predicted identical growth rates for all 81 knockout strains under both media. MDFBA, on the other hand, predicted different growth rates for 67 of the knockout strains under the two growth media, correctly predicting a higher growth rate in NacetylDglucosamine in 87% of these cases.
Extending the gene essentiality analysis under these media for other genes, not included in the ASAP dataset, revealed several additional scenarios in which MDFBA and FBA predictions significantly differ. We found that, generally, MDFBA predicts the activation of reactions involved in cofactor biosynthesis that are not activated by FBA (the distribution of reactions whose predicted activity pattern differ between MDFBA and FBA across various metabolic subsystems is shown in Additional file 3). For example, under succinate minimal medium, MDFBA predicts that genes in the ubiquinone8 biosynthetic pathway are essential for growth, whereas FBA predicts these genes to be nonessential (Figure 3). Specifically, both methods predict that the first part of this pathway, leading to the production of the biomass metabolite 2octaprenyl6hydroxyphenol (black solid edges), is essential under succinate minimal medium, while only MDFBA predicts that the remaining part of the pathway, leading to ubiquinone8, is activated. Ubiquinone8 is an important redox cofactor in E. coli's aerobic respiration, switching between a reduced (q8h2) state and an oxidized (q8) state. While both FBA and MDFBA predict the cycling of ubiquinone8 between the reduced and oxidized states under succinate minimal medium (as part of aerobic respiration), only MDFBA predicts the corresponding requirement for de novo synthesis of this metabolite to accommodate for its growthassociated dilution. Notably, this scenario is similar to that described in the toy model in Figure 1a, where q8 and q8h2 correspond to cofactor metabolites C and C*. As a testimony to the correctness of these predictions, we found that a gene coding for an enzyme catalyzing two reactions in the ubiquinone8 biosynthetic pathway, ubiG, was experimentally validated to be essential for E. coli growing under succinate minimal medium [30, 31]. Adding ubiquinone8 to the biomass reaction would indeed solve the false essentiality prediction of ubiG under succinate minimal medium, but would lead to a false essentiality prediction under glucose medium, where ubiG was shown to be nonessential for growth [32]  further emphasizing the advantage of accounting for metabolite dilution in FBA.
As an additional validation, we applied MDFBA to predict gene essentiality for 1,117 genes under glucose and glycerol minimal media, based on measurements from [32] and [33], respectively. Each gene in the dataset was experimentally determined to be either essential or nonessential and the accuracy of the essentiality predictions obtained by FBA and MDFBA was assessed via an area under curve (AUC) score of the receiver operating characteristic (ROC) curve [34]. This curve represents the true positive and false positive rates as a function of the threshold on the predicted growth rate used to determine gene essentiality (experimental and predicted datasets are available in Additional file 4). Initially, we applied MDFBA, utilizing the same definition of a biomass as in FBA (as performed above), obtaining very similar AUC scores of 0.888/0.873 and 0.873/0.875 for MDFBA and FBA, respectively, under glucose/glycerol. However, following further inspection, we found that 15 of the 63 metabolic precursors that make up the biomass are actually designated as cofactors by Feist et al. [6]; hence, MDFBA is likely to be able to predict the growthassociated demand for their synthesis specifically under the conditions in which they are required, without accounting for them explicitly in the biomass definition. For example, in Figure 1, the dilution of cofactor C is correctly predicted by MDFBA in a contextdependent manner only when metabolite X is present in the medium, as C is not fixed to be included in the biomass. Falsely including metabolite C in the biomass, although it is required in only some media, would lead to a false prediction of lethality when metabolite X is absent from the growth medium. Given that such an inclusion of cofactors in the biomass may lead to false gene essentiality predictions, their removal from the biomass is likely to improve prediction performance. In order to remove these cofactors from the biomass, we performed the following preprocessing step: in each growth condition examined, each cofactor was in turn removed from the biomass and MDFBA was then applied to test whether a dilution is predicted for the cofactor under a subset of the gene knockout strains. The analysis revealed three cofactors (10formyltetrahydrofolate, 2octaprenyl6hydroxyphenol, flavin adenine dinucleotide oxidized (FAD)) whose dilution is dynamically predicted by MDFBA and they were subsequently removed from MDFBA's biomass (dilution analysis results are available in Additional file 5). Repeating the gene essentiality analysis with the reduced biomass considerably improved the prediction performance of MDFBA (Figure 4). Specifically, the AUC scores achieved by MDFBA and FBA under glucose medium are 0.910 and 0.873, respectively, and under glycerol medium are 0.893 and 0.875, respectively. As further support for the assertion that the improved prediction performance is not a mere consequence of removing unnecessary biomass precursors, we reapplied FBA using the same reduced biomass (labeled FBAr in Figure 4), which showed no improvement over FBA's original performance. These results clearly demonstrate the addedvalue of considering the contextdependent nature of cofactor requirements, which can change depending on both genetic and environmental factors.
Discussion
This study presents MDFBA, a variant of FBA for predicting metabolic flux distributions by accounting for growthassociated dilution of all metabolites in a contextdependent manner. The method predicts feasible flux distributions maximizing the production rate of a predefined biomass while accounting for the dilution of all intermediate metabolites, and most importantly, for all metabolic cofactors involved in the process. MDFBA was shown to successfully predict E. coli's gene essentiality under a variety of growth media and knockout strains, displaying a significant improvement upon the prediction performance of the commonly used FBA method.
MDFBA has two notable limitations, which may contribute to the relatively low improvement in growth rate prediction accuracy (compared to the marked advantage in predicting gene knockout lethality). First, MDFBA employs a uniform lower bound on the dilution rate of intermediate metabolites which, along with the absence of reactions outside the scope of the network model that degrade intermediate metabolites, implicitly reflects the assumption of a uniform concentration of all intermediate metabolites. A natural extension of MDFBA would be to consider different lower and upper bounds on concentrations of different metabolites, based on concentration statistics gathered via metabolomic measurements across a variety of conditions (for example, [24]). Notably though, changing the lower bound employed here to a range of possible values and incorporating an upper bound on dilution rates across all metabolites did not improve the prediction performance (data not shown). Second, MDFBA, similarly to FBA, is based on the assumption that microbial species aim to maximize their growth rate and hence search for feasible flux distributions that maximize biomass synthesis rate. However, previous studies have questioned this hypothesis, suggesting alternative possible optimization criteria. Future studies should investigate the potential usage of such optimization criteria with MDFBA [35]. More generally, CBM methods that do not rely on optimization may also benefit from variants that account for metabolite dilution during growth.
A marked disadvantage of MDFBA is its dependence on MILP, which is computationally more demanding than LP, utilized by FBA. To improve the runtime of MDFBA, the amount of integer variables in the MDFBA formulation may be reduced by employing a previous method to identify the metabolic 'scope' of the medium nutrients. Specifically, Handorf et al. [36] investigated the capacity to produce metabolites from available medium nutrients by applying FBA and a network expansion algorithm, resulting in a production scope for each set of medium metabolites. A potential improvement in runtime may be achieved by calculating the scope of the input growth medium and assigning integer variables only for metabolites in that derived scope, as all the other metabolites will never be able to satisfy their dilution demand. Speeding up the runtime may be of importance when applying MDFBA to larger networks, such as the recently published human model [16], or when probing the network under multiple knockout configurations [37, 38].
An interesting comparison can be made between MDFBA and a method developed by Price et al. [39] for eliminating futile cycles via the identification of type III extreme pathways (that is, a unique set of convex basis vectors of the flux distribution solution space that do not include exchange reactions). While the extreme pathways method enables the elimination of thermodynamically impossible loops, MDFBA removes infeasible solutions due to dilution demands. Notably, the latter method also implicitly eliminates type III extreme pathways since these pathways do not satisfy dilution demands of the participating metabolites. Additionally, MDFBA eliminates solutions that do not involve type III extreme pathways as demonstrated in Figure 1b: when metabolite X is absent from the growth medium, the cycle involving reactions v_{4} and v_{8} cannot be activated based on MDFBA, since the dilution of cofactor C cannot be satisfied, although this cycle is not part of a type III extreme pathway.
Another appealing application of MDFBA could be the identification of missing reactions in the model by comparing predicted phenotypes with measured ones, in line with previous works using FBA for this purpose [40]. For example, suppose that in Figure 1a the biosynthetic pathway for metabolite C, through reactions v_{6} and v_{7}, was not included in the model. In this case, MDFBA would predict metabolic flow through reactions v_{2} and v_{3}, such that the enzymes catalyzing these reactions are essential, contrary to experimental essentiality data. Utilizing a method similar to that used by Reed et al. [40], using MDFBA can infer the missing reactions, v_{6} and v_{7}. Employing FBA for this purpose would not work since FBA predicts v_{2} and v_{3} to be nonessential, as the activity of reactions v_{4} and v_{8} do not depend on the presence of reactions v_{6} and v_{7}.
While this work applied MDFBA to predict metabolic phenotypes in E. coli, for which a comprehensive and accurate metabolic network model exists, the method can also be applied to any one of a growing number of reconstructed network models [20]. Importantly, the application of MDFBA to other network models is straightforward and requires no modelspecific data curation. To facilitate simple usage of MDFBA, we provide an implementation of the method in the supplemental website [41]. A particularly interesting potential application of MDFBA would be for modeling malignant proliferating cells in human cancer, potentially revealing the activity of biosynthetic pathways for various cofactors required to balance their growthassociated dilution. The latter may utilize the recently published model of human cellular metabolism by [16] or [42]. Overall, we expect that future use of MDFBA will promote improved metabolic phenotypic predictions across a variety of organisms, growth conditions and genetic alterations.
Materials and methods
Metabolite dilution flux balance analysis
To formulate a massbalance constraint while accounting for metabolite growth dilution, we assume that each metabolite j that is produced by a certain reaction at a rate greater than zero (referred to as an 'active metabolite') has a nonzero concentration and should hence be diluted with a rate greater than zero (denoted by d_{ j }). To compute a feasible flux distribution, , and a corresponding vector of dilution rates, , we employ the following optimization problem:
subject to
where a massbalance constraint, accounting for the dilution of all active metabolites, is formulated in Equation 3. Equation 4 assigns a positive dilution rate above a predefined threshold (denoted by Îµ) for active metabolites, produced in some nonzero rate in the flux distribution . In our application of the method for E. coli we set Îµ = 10^{4} Î¼mol/mg, which represents a common concentration of intermediate metabolites [6]. Notably, the model's predictions were robust to different choices of Îµ values (data not shown). Enzyme directionality and capacity constraints are formulated in Equation 5 by imposing and as lower and upper bounds on flux values.
The above optimization problem is solved by formulating it as a MILP problem, replacing the Equation 4 constraint with the linear equations specified below: for each metabolite j in the model, we define an integer variable y_{ j }that denotes whether the metabolite is active (that is, being produced by some nonzero reaction in the model), via the following linear constraints:
where R_{ j }denotes the set of reactions in which metabolite j participates. Equation 6 is a linear formulation of the statement 'if Î½_{ i }â‰¥ Îµ then y_{ j }= 1' and Equation 7 is the symmetric for negative fluxes (that is, Î½_{ i }â‰¤ Îµ). Given the variables, Equation 4 can be formulated via the following constraints:
which can be represented in linear form (since ÎµÎ¼ < 1) as:
A simplified formulation assuming a constant growth rate of Î¼ = 1 in Equation 8 (for calculating the dilution rate of intermediate metabolites) gave qualitatively similar results to the above linear formulation (data not shown). The commercial solver CPLEX running on 64bit Linux machines was used for solving LP and MILP problems within a few dozens of seconds per problem.
Abbreviations
 AUC:

area under curve
 CBM:

constraintbased modeling
 FBA:

flux balance analysis
 LP:

linear programming
 MDFBA:

metabolite dilution flux balance analysis
 MILP:

mixed integer linear programming
 OD:

optical density
 q8:

ubiquinone8oxidized
 q8h2:

ubiquinone8reduced
 ROC:

receiver operating characteristic.
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Acknowledgements
We are grateful to Hadas Zur, Naama Tepper and Yoav Teboulle for their fruitful comments. This study was supported in part by a fellowship from the Edmond J Safra Bioinformatics program at TelAviv University. ER's and TS's research is supported by grants from the Israel Science Foundation.
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Authors' contributions
TB developed the method, implemented the method, performed analyses and wrote the manuscript. OF performed analyses. ER conceived the study. TS conceived the study, developed the method and wrote the manuscript. All authors discussed the results, and read, commented and approved the final manuscript.
Electronic supplementary material
13059_2009_2336_MOESM1_ESM.xlsx
Additional file 1: ASAP growth conditions. Excel file including the growth conditions used to model the ASAP experiments used by both FBA and MDFBA. (XLSX 15 KB)
13059_2009_2336_MOESM2_ESM.xlsx
Additional file 2: ASAP experimental optical density values. Excel file including the experimentally measured OD values taken from the ASAP database. (XLSX 64 KB)
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Additional file 3: Reaction activity difference across subsystems. Figure showing the mean reaction activity difference between FBA and MDFBA across the different metabolic subsystems available in the model. (XLSX 59 KB)
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Additional file 4: Gene essentiality analysis results. Excel file containing the gene essentiality predictions by FBA and MDFBA as well as the experimental gene essentiality data. (XLSX 198 KB)
13059_2009_2336_MOESM5_ESM.xlsx
Additional file 5: Cofactor dilutionrelated demand activity predictions. Excel file containing MDFBA's predictions for the dilution related growthdemand synthesis of the three cofactors: 10formyltetrahydrofolate (10fthf), 2octaprenyl6hydroxyphenol (2ohph) and FAD across different gene knockouts. (XLSX 52 KB)
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Benyamini, T., Folger, O., Ruppin, E. et al. Flux balance analysis accounting for metabolite dilution. Genome Biol 11, R43 (2010). https://doi.org/10.1186/gb2010114r43
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DOI: https://doi.org/10.1186/gb2010114r43