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Retrieval and visualization of biological data are essential for understanding complex systems. With the increasing volume of data generated from high-throughput sequencing technologies, effective and optimized data visualization tools have become indispensable. This is particularly relevant in the COVID-19 postpandemic period, where understanding the diversity and interactions of microbial communities (i.e., viral and bacterial) constitutes an important asset to develop and plan suitable interventions.In this chapter, we show the usage and the potentials of ExTaxsI (Exploring Taxonomy Information) tool to retrieve viral biodiversity data stored in National Center for Biotechnology Information (NCBI) databases and create the related visualization. In addition, by integrating different functions and modules, the tool generates relevant types of visualization plots to facilitate the exploration of microbial biodiversity communities useful to deep dive into ecological and taxonomic relationships among different species and identify potential significant targets.Using the Monkeypox virus as a case study, this work points out significant perspectives on biological data visualization, which can be used to gain insights into the ecology, evolution, and pathogenesis of viruses. Accordingly, we show the potentiality of ExTaxsI to organize and describe the available/downloaded data in an easy, simple, and interpretable way allowing the user to interact dynamically with the visualization plots through specific filters, zoom, and explore functions.

Metabolism is directly and indirectly fine-tuned by a complex web of interacting regulatory mechanisms that fall into two major classes. On the one hand, the expression level of the catalyzing enzyme sets the maximal theoretical flux level (i.e., the net rate of the reaction) for each enzyme-controlled reaction. On the other hand, metabolic regulation controls the metabolic flux through the interactions of metabolites (substrates, cofactors, allosteric modulators) with the responsible enzyme. High-throughput data, such as metabolomics and transcriptomics data, if analyzed separately, do not accurately characterize the hierarchical regulation of metabolism outlined above. They must be integrated to disassemble the interdependence between different regulatory layers controlling metabolism. To this aim, we propose INTEGRATE, a computational pipeline that integrates metabolomics and transcriptomics data, using constraint-based stoichiometric metabolic models as a scaffold. We compute differential reaction expression from transcriptomics data and use constraintbased modeling to predict if the differential expression of metabolic enzymes directly originates differences in metabolic fluxes. In parallel, we use metabolomics to predict how differences in substrate availability translate into differences in metabolic fluxes. We discriminate fluxes regulated at the metabolic and/or gene expression level by intersecting these two output datasets. We demonstrate the pipeline using a set of immortalized normal and cancer breast cell lines. In a clinical setting, knowing the regulatory level at which a given metabolic reaction is controlled will be valuable to inform targeted, truly personalized therapies in cancer patients.

Plastic has become an indispensable material in many fields, with production increasing every year; however, most of the plastic waste is still incinerated or landfilled, and only 10% of the new plastic is recycled even once. Considering that plastic is derived from petroleum-based resources, this creates a worrying loss of resources and a cascade of environmental issues. Among all plastic, polyethylene terephthalate (PET) is the most produced polyester worldwide (56 Mt/year). This work focuses on the upcycling of PET monomers – terephthalic acid (TPA) and ethylene glycol (EG) – by yeast fermentation for the production of industrially relevant organic acids (protocatechuic acid, cis,cis-muconic acid, 3-carboxy-cis,cis-muconic acid, glycolic acid). Thanks to synthetic biology (EASY-MISE toolkit) and metabolic engineering, different Saccharomyces cerevisiae strains were created for the bioconversion of TPA and the use of EG as a carbon source. For the bioconversion of TPA, several genes from Ideonella sakaiensis were introduced; TPA toxicity on the new strains was assessed in microwell plates. Since EG assimilation has never been described in yeast, different medium combinations were tested for the assimilation of EG; in parallel, two synthetic metabolic pathways were introduced in S. cerevisiae for a more efficient assimilation. In silico constraint-based models are currently being developed to optimize growth and production on TPA and EG, and to shed light on the yeast native metabolism of the latter. Our research shows promising results in the biodegradation and upcycling of PET monomers by yeast fermentation, and it will become even more interesting thanks to the recent advances in enzymatic PET hydrolysis.

Background: Genome-wide reconstructions of metabolism opened the way to thorough investigations of cell metabolism for health care and industrial purposes. However, the predictions offered by Flux Balance Analysis (FBA) can be strongly affected by the choice of flux boundaries, with particular regard to the flux of reactions that sink nutrients into the system. To mitigate possible errors introduced by a poor selection of such boundaries, a rational approach suggests to focus the modeling efforts on the pivotal ones. Methods: In this work, we present a methodology for the automatic identification of the key fluxes in genome-wide constraint-based models, by means of variance-based sensitivity analysis. The goal is to identify the parameters for which a small perturbation entails a large variation of the model outcomes, also referred to as sensitive parameters. Due to the high number of FBA simulations that are necessary to assess sensitivity coefficients on genome-wide models, our method exploits a master-slave methodology that distributes the computation on massively multi-core architectures. We performed the following steps: (1) we determined the putative parameterizations of the genome-wide metabolic constraint-based model, using Saltelli’s method; (2) we applied FBA to each parameterized model, distributing the massive amount of calculations over multiple nodes by means of MPI; (3) we then recollected and exploited the results of all FBA runs to assess a global sensitivity analysis. Results: We show a proof-of-concept of our approach on latest genome-wide reconstructions of human metabolism Recon2.2 and Recon3D. We report that most sensitive parameters are mainly associated with the intake of essential amino acids in Recon2.2, whereas in Recon 3D they are associated largely with phospholipids. We also illustrate that in most cases there is a significant contribution of higher order effects. Conclusion: Our results indicate that interaction effects between differe

Metabolic network models are increasingly being used in health care and industry. As a consequence, many tools have been released to automate their reconstruction process de novo. In order to enable gene deletion simulations and integration of gene expression data, these networks must include gene-protein-reaction (GPR) rules, which describe with a Boolean logic relationships between the gene products (e.g., enzyme isoforms or subunits) associated with the catalysis of a given reaction. Nevertheless, the reconstruction of GPRs still remains a largely manual and time consuming process. Aiming at fully automating the reconstruction process of GPRs for any organism, we propose the open-source python-based framework GPRuler. By mining text and data from 9 different biological databases, GPRuler can reconstruct GPRs starting either from just the name of the target organism or from an existing metabolic model. The performance of the developed tool is evaluated at small-scale level for a manually curated metabolic model, and at genome-scale level for three metabolic models related to Homo sapiens and Saccharomyces cerevisiae organisms. By exploiting these models as benchmarks, the proposed tool shown its ability to reproduce the original GPR rules with a high level of accuracy. In all the tested scenarios, after a manual investigation of the mismatches between the rules proposed by GPRuler and the original ones, the proposed approach revealed to be in many cases more accurate than the original models. By complementing existing tools for metabolic network reconstruction with the possibility to reconstruct GPRs quickly and with a few resources, GPRuler paves the way to the study of context-specific metabolic networks, representing the active portion of the complete network in given conditions, for organisms of industrial or biomedical interest that have not been characterized metabolically yet.

Laboratory models derived from clinical samples represent a solid platform in preclinical research for drug testing and investigation of disease mechanisms. The integration of these laboratory models with their digital counterparts (i.e., predictive mathematical models) allows to set up digital twins essential to fully exploit their potential to face the enormous molecular complexity of human organisms. In particular, due to the close integration of cell metabolism with all other cellular processes, any perturbation in cellular physiology typically reflect on altered cells metabolic profiling. In this regard, changes in metabolism have been shown, also in our laboratory, to drive a causal role in the emergence of cancer disease. Nevertheless, a unique metabolic program does not describe the altered metabolic profile of all tumour cells due to many causes from genetic variability to intratumour heterogeneous dependency on nutrients consumption and metabolism by multiple co-existing subclones. Currently, fluxomics approaches just match with the necessity of characterizing the overall flux distribution of cells within given samples, by disregarding possible heterogeneous behaviors. For the purpose of stratifying cancer heterogeneous subpopulations, quantification of fluxes at the single-cell level is needed. To this aim, we here present a new computational framework called single-cell Flux Balance Analysis (scFBA) that aims to set up digital metabolic twins in the perspective of being better exploited within a framework that makes also use of laboratory patient cell models. In particular, scFBA aims at integrating single-cell RNA-seq data within computational population models in order to depict a snapshot of the corresponding single-cell metabolic phenotypes at a given moment, together with an unsupervised identification of metabolic subpopulations.

FBCA (Flux Balance Cellular Automata) has been recently proposed as a new multi-scale modeling framework to represent the spatial dynamics of multi-cellular systems, while simultaneously taking into account the metabolic activity of individual cells. Preliminary results have revealed the potentialities of the framework in enabling to identify and analyze complex emergent properties of cellular populations, such as spatial patterns phenomena and synchronization effects. Here we move a step forward, by exploring the possibility of integrating real-world data into the framework. To this end, we seek to customize the metabolism of individual cells according to single-cell gene expression profiles. We investigate the effect on cell metabolism of the interplay between: (a) the environmental conditions determined by nutrient diffusion dynamics; (b) the activation or deactivation of metabolic pathways determined by gene expression.

Boosted by the exponential growth of microbiome-based studies, analyzing microbiome patterns is now a hot-topic, finding different fields of application. In particular, the use of machine learning techniques is increasing in microbiome studies, providing deep insights into microbial community composition. In this context, in order to investigate microbial patterns from 16S rRNA metabarcoding data, we explored the effectiveness of Association Rule Mining (ARM) technique, a supervised-machine learning procedure, to extract patterns (in this work, intended as groups of species or taxa) from microbiome data. ARM can generate huge amounts of data, making spurious information removal and visualizing results challenging. Our work sheds light on the strengths and weaknesses of pattern mining strategy into the study of microbial patterns, in particular from 16S rRNA microbiome datasets, applying ARM on real case studies and providing guidelines for future usage. Our results highlighted issues related to the type of input and the use of metadata in microbial pattern extraction, identifying the key steps that must be considered to apply ARM consciously on 16S rRNA microbiome data. To promote the use of ARM and the visualization of microbiome patterns, specifically, we developed microFIM (microbial Frequent Itemset Mining), a versatile Python tool that facilitates the use of ARM integrating common microbiome outputs, such as taxa tables. microFIM implements interest measures to remove spurious information and merges the results of ARM analysis with the common microbiome outputs, providing similar microbiome strategies that help scientists to integrate ARM in microbiome applications. With this work, we aimed at creating a bridge between microbial ecology researchers and ARM technique, making researchers aware about the strength and weaknesses of association rule mining approach.

Background: The increasing availability of multi-omics data is leading to regularly revised estimates of existing biodiversity data. In particular, the molecular data enable novel species to be characterized and the information linked to those already observed to be increased with new genomics data. For this reason, the management and visualization of existing molecular data, and their related metadata, through the implementation of easy-to-use IT tools have become a key point to design future research. The more users are able to access biodiversity-related information, the greater the ability of the scientific community to expand its knowledge in this area. Results: In this article we focus on the development of ExTaxsI (Exploring Taxonomy Information), an IT tool that can retrieve biodiversity data stored in NCBI databases and provide a simple and explorable visualization. We use 3 case studies to show how an efficient organization of the available data can lead to obtaining new information that is fundamental as a starting point for new research. Using this approach highlights the limits in the distribution of data availability, a key factor to consider in the experimental design phase of broad-spectrum studies such as metagenomics. Conclusions: ExTaxsI can easily retrieve molecular data and its metadata with an explorable visualization, with the aim of helping researchers to improve experimental designs and highlight the main gaps in the coverage of available data.

Background: Over the past few years, studies have increasingly focused on the development of mobile apps as complementary tools to existing traditional pharmacovigilance surveillance systems for improving and facilitating adverse drug reaction (ADR) reporting. Objective: In this research, we evaluated the potentiality of a new mobile app (vaxEffect@UniMiB) to perform longitudinal studies, while preserving the anonymity of the respondents. We applied the app to monitor the ADRs during the COVID-19 vaccination campaign in a sample of the Italian population. Methods: We administered vaxEffect@UniMiB to a convenience sample of academic subjects vaccinated at the Milano-Bicocca University hub for COVID-19 during the Italian national vaccination campaign. vaxEffect@UniMiB was developed for both Android and iOS devices. The mobile app asks users to send their medical history and, upon every vaccine administration, their vaccination data and the ADRs that occurred within 7 days postvaccination, making it possible to follow the ADR dynamics for each respondent. The app sends data over the web to an application server. The server, along with receiving all user data, saves the data in a SQL database server and reminds patients to submit vaccine and ADR data by push notifications sent to the mobile app through Firebase Cloud Messaging (FCM). On initial startup of the app, a unique user identifier (UUID) was generated for each respondent, so its anonymity was completely ensured, while enabling longitudinal studies. Results: A total of 3712 people were vaccinated during the first vaccination wave. A total of 2733 (73.6%) respondents between the ages of 19 and 80 years, coming from the University of Milano-Bicocca (UniMiB) and the Politecnico of Milan (PoliMi), participated in the survey. Overall, we collected information about vaccination and ADRs to the first vaccine dose for 2226 subjects (60.0% of the first dose vaccinated), to the second dose for 1610 subjects (43.4% of the s

Background: Determining the value of kinetic constants for a metabolic system in the exact physiological conditions is an extremely hard task. However, this kind of information is of pivotal relevance to effectively simulate a biological phenomenon as complex as metabolism. Results: To overcome this issue, we propose to investigate emerging properties of ensembles of sets of kinetic constants leading to the biological readout observed in different experimental conditions. To this aim, we exploit information retrievable from constraint-based analyses (i.e. metabolic flux distributions at steady state) with the goal to generate feasible values for kinetic constants exploiting the mass action law. The sets retrieved from the previous step will be used to parametrize a mechanistic model whose simulation will be performed to reconstruct the dynamics of the system (until reaching the metabolic steady state) for each experimental condition. Every parametrization that is in accordance with the expected metabolic phenotype is collected in an ensemble whose features are analyzed to determine the emergence of properties of a phenotype. In this work we apply the proposed approach to identify ensembles of kinetic parameters for five metabolic phenotypes of E. Coli, by analyzing five different experimental conditions associated with the ECC2comp model recently published by Hädicke and collaborators. Conclusions: Our results suggest that the parameter values of just few reactions are responsible for the emergence of a metabolic phenotype. Notably, in contrast with constraint-based approaches such as Flux Balance Analysis, the methodology used in this paper does not require to assume that metabolism is optimizing towards a specific goal.

We present MaREA4Galaxy, a user-friendly tool that allows a user to characterize and to graphically compare groups of samples with different transcriptional regulation of metabolism, as estimated from cross-sectional RNA-seq data. The tool is available as plug-in for the widely-used Galaxy platform for comparative genomics and bioinformatics analyses. MaREA4Galaxy combines three modules. The Expression2RAS module, which, for each reaction of a specified set, computes a Reaction Activity Score (RAS) as a function of the expression level of genes encoding for the associated enzyme. The MaREA (Metabolic Reaction Enrichment Analysis) module that allows to highlight significant differences in reaction activities between specified groups of samples. The Clustering module which employs the RAS computed before as a metric for unsupervised clustering of samples into distinct metabolic subgroups; the Clustering tool provides different clustering techniques and implements standard methods to evaluate the goodness of the results.

In the latter years, detailed genome-wide metabolic models have been proposed, paving the way to thorough investigations of the connection between genotype and phenotype in human cells. Nevertheless, classic modeling and dynamic simulation approaches—based either on differential equations integration, Markov chains or hybrid methods—are still unfeasible on genome-wide models due to the lack of detailed information about kinetic parameters and initial molecular amounts. By relying on a steady-state assumption and constraints on extracellular fluxes, constraint-based modeling provides an alternative means—computationally less expensive than dynamic simulation—for the investigation of genome-wide biochemical models. Still, the predictions provided by constraint-based analysis methods (e.g., flux balance analysis) are strongly dependent on the choice of flux boundaries. To contain possible errors induced by erroneous boundary choices, a rational approach suggests to focus on the pivotal ones. In this work we propose a novel methodology for the automatic identification of the key fluxes in large-scale constraint-based models, exploiting variance-based sensitivity analysis and distributing the computation on massively multi-core architectures. We show a proof-of-concept of our approach on core models of relatively small size (up to 314 reactions and 256 chemical species), highlighting the computational challenges.

Motivation: Intratumour heterogeneity poses many challenges to the treatment of cancer. Unfortunately, the transcriptional and metabolic information retrieved by currently available computational and experimental techniques portrays the average behaviour of intermixed and heterogeneous cell subpopulations within a given tumour. Emerging single-cell genomic analyses are nonetheless unable to characterize the interactions among cancer subpopulations. In this study, we propose popFBA, an extension to classic Flux Balance Analysis, to explore how metabolic heterogeneity and cooperation phenomena affect the overall growth of cancer cell populations. Results: We show how clones of a metabolic network of human central carbon metabolism, sharing the same stoichiometry and capacity constraints, may follow several different metabolic paths and cooperate to maximize the growth of the total population. We also introduce a method to explore the space of possible interactions, given some constraints on plasma supply of nutrients. We illustrate how alternative nutrients in plasma supply and/or a dishomogeneous distribution of oxygen provision may affect the landscape of heterogeneous phenotypes. We finally provide a technique to identify the most proliferative cells within the heterogeneous population.

Metabolic reprogramming is a general feature of cancer cells. Regrettably, the comprehensive quantification of metabolites in biological specimens does not promptly translate into knowledge on the utilization of metabolic pathways. By estimating fluxes across metabolic pathways, computational models hold the promise to bridge this gap between data and biological functionality. These models currently portray the average behavior of cell populations however, masking the inherent heterogeneity that is part and parcel of tumorigenesis as much as drug resistance. To remove this limitation, we propose single-cell Flux Balance Analysis (scFBA) as a computational framework to translate single-cell transcriptomes into single-cell fluxomes. We show that the integration of single-cell RNA-seq profiles of cells derived from lung adenocarcinoma and breast cancer patients into a multi-scale stoichiometric model of a cancer cell population: significantly 1) reduces the space of feasible single-cell fluxomes; 2) allows to identify clusters of cells with different growth rates within the population; 3) points out the possible metabolic interactions among cells via exchange of metabolites. The scFBA suite of MATLAB functions is available at https://github.com/ BIMIB-DISCo/scFBA, as well as the case study datasets.

Understanding the synchronization, either induced or spontaneous, of cell growth, division and proliferation in a cell culture is an important topic in molecular biology and biotechnology. Metabolic processes related to the synthesis of all the molecules needed for a new round of cell division are the basic underlying phenomena responsible for the behaviour of the population. Complex dynamics, such as population oscillations, arise when the individual members of a population divide in unison. To investigate the conditions that can determine oscillatory behaviors, we here use a multi-scale model that couples the simulation of metabolic growth, via metabolic network modelling and Flux Balance Analysis, with the simulation of population and spatial dynamics, via Cellular Potts Models. We here show that repeated oscillations in the overall number of cells spontaneously emerge, due to the synchronization of duplication events, unless cell density-dependent controls on growth are introduced

The metabolic processes related to the synthesis of the molecules needed for a new round of cell division underlie the complex behaviour of cell populations in multi-cellular systems, such as tissues and organs, whereas their deregulation can lead to pathological states, such as cancer. Even within genetically homogeneous populations, complex dynamics, such as population oscillations or the emergence of specific metabolic and/or proliferative patterns, may arise, and this aspect is highly amplified in systems characterized by extreme heterogeneity. To investigate the conditions and mechanisms that link metabolic processes to cell population dynamics, we here employ a previously introduced multi-scale model of multi-cellular system, named FBCA (Flux Balance Analysis with Cellular Automata), which couples biomass accumulation, simulated via Flux Balance Analysis of a metabolic network, with the simulation of population and spatial dynamics via Cellular Potts Models. In this work, we investigate the influence that different modes of nutrients diffusion within the system may have on the emerging behaviour of cell populations. In our model, metabolic communication among cells is allowed by letting secreted metabolites to diffuse over the lattice, in addition to diffusion of nutrients from given sources. The inclusion of the diffusion processes in the model proved its effectiveness in characterizing plausible biological scenarios.

Cancer cells share several metabolic traits, including aerobic production of lactate from glucose (Warburg effect), extensive glutamine utilization and impaired mitochondrial electron flow. It is still unclear how these metabolic rearrangements, which may involve different molecular events in different cells, contribute to a selective advantage for cancer cell proliferation. To ascertain which metabolic pathways are used to convert glucose and glutamine to balanced energy and biomass production, we performed systematic constraint-based simulations of a model of human central metabolism. Sampling of the feasible flux space allowed us to obtain a large number of randomly mutated cells simulated at different glutamine and glucose uptake rates. We observed that, in the limited subset of proliferating cells, most displayed fermentation of glucose to lactate in the presence of oxygen. At high utilization rates of glutamine, oxidative utilization of glucose was decreased, while the production of lactate from glutamine was enhanced. This emergent phenotype was observed only when the available carbon exceeded the amount that could be fully oxidized by the available oxygen. Under the latter conditions, standard Flux Balance Analysis indicated that: this metabolic pattern is optimal to maximize biomass and ATP production; it requires the activity of a branched TCA cycle, in which glutamine-dependent reductive carboxylation cooperates to the production of lipids and proteins; it is sustained by a variety of redox-controlled metabolic reactions. In a K-ras transformed cell line we experimentally assessed glutamine-induced metabolic changes. We validated computational results through an extension of Flux Balance Analysis that allows prediction of metabolite variations. Taken together these findings offer new understanding of the logic of the metabolic reprogramming that underlies cancer cell growth.

Real-world evidence can provide answers on healthcare utilization and appropriateness, post-marketing drugs safety and comparative effectiveness, and cost-effectiveness profiles of healthcare pathways. Healthcare utilization databases, possibly integrated with drug and disease registries, electronic medical records, survey and cohort data (i.e. real-world data), allow to trace healthcare ‘footprints’ left from beneficiaries of National Health Service. Beaver is a research platform available on demand to Italian regions which we developed for computing indicators of healthcare utilization and clinical outcomes, as well as for generating evidence on effectiveness and cost-effectiveness profile. Two distinct solutions may be adopted. One, the so-called Beaver Light front-end allows to automatically compute health indicators of adherence to official guidelines. Two, the so-called Beaver Full front-end involves an eight-step procedure entirely driven by the study protocol. In order to fulfil the directives recently issued by the European Parliament and Council and the Italian Authority for the protection of individual data, the platform resides in each region’s infrastructure, so limiting the free movement of electronic health data. Indeed, regional authorities should be responsible for data safety and for allowing data accessibility. The use of standardized and validated algorithms enables to obtain regional estimates that, being obtained by employing regional platforms containing data extracted with standardized procedure, may be compared and possibly summarized by using common meta-analytic techniques. In conclusion, the Beaver regional platform is a promising tool which may contribute to stimulate healthcare research in Italy.

Constraint-based approaches have been proven useful to determine steady state fluxes in metabolic models, however they are not able to determine metabolite concentrations and they imply the assumption that a biological process is optimized towards a given function. In this work we define a computational strategy exploiting mechanism based simulations as a framework to determine, through a filtering procedure, ensembles of kinetic constants and steady state metabolic concentrations that are in agreement with one or more metabolic phenotypes, avoiding at the same time the need of assuming an optimization mechanism. To test our procedure we exploited a model of yeast metabolism and we filtered trajectories accordingly to a loose definition of the Crabtree phenotype.

The intratumor heterogeneity has been recognized to characterize cancer cells impairing the efficacy of cancer treatments. We here propose an extension of constraint-based modeling approach in order to simulate metabolism of cell populations with the aim to provide a more complete characterization of these systems, especially focusing on the relationships among their components. We tested our methodology by using a toy-model and taking into account the main metabolic pathways involved in cancer metabolic rewiring. This toy-model is used as “individual” to construct a “population model” characterized by multiple interacting individuals, all having the same topology and stoichiometry, and sharing the same nutrients supply. We observed that, in our population, cancer cells cooperate with each other to reach a common objective, but without necessarily having the same metabolic traits. We also noticed that the heterogeneity emerging from the population model is due to the mismatch between the objective of the individual members and the objective of the entire population.

The links between metabolic dysfunctions and various diseases or pathological conditions are being increasingly revealed. This revival of interest in cellular metabolism has pushed forward new experimental technologies enabling the characterization of metabolic phenotypes. Unfortunately, while large datasets are being collected, which encompass the concentration of many metabolites of a system under different conditions, these datasets remain largely obscure. In fact, in spite of the efforts to interpret alterations in metabolic concentrations, it is difficult to correctly ascribe them to the corresponding variations in metabolic fluxes (i.e. the rate of turnover of molecules through metabolic pathways) and thus to the up- or down-regulation of given pathways. As a first step towards a systematic procedure to connect alterations in metabolic fluxes with shifts in metabolites, we propose to exploit a Montecarlo approach to look for correlations between the variations in fluxes and in metabolites, observed when simulating the response of a metabolic network to a given perturbation. As a proof of principle, we investigate the dynamics of a simplified ODE model of yeast metabolism under different glucose abundances. We show that, although some linear correlations between shifts in metabolites and fluxes exist, those relationships are far from obvious. In particular, metabolite levels can show a low correlation with changes in the fluxes of the reactions that directly involve them, while exhibiting a strong connection with alterations in fluxes that are far apart in the network.

The metabolic rearrangements occurring in cancer cells can be effectively investigated with a Systems Biology approach supported by metabolic network modeling. We here present tissue-specific constraint-based core models for three different types of tumors (liver, breast and lung) that serve this purpose. The core models were extracted and manually curated from the corresponding genome-scale metabolic models in the Human Metabolic Atlas database with a focus on the pathways that are known to play a key role in cancer growth and proliferation. Along similar lines, we also reconstructed a core model from the original general human metabolic network to be used as a reference model. A comparative Flux Balance Analysis between the reference and the cancer models highlighted both a clear distinction between the two conditions and a heterogeneity within the three different cancer types in terms of metabolic flux distribution. These results emphasize the need for modeling approaches able to keep up with this tumoral heterogeneity in order to identify more suitable drug targets and develop effective treatments. According to this perspective, we identified key points able to reverse the tumoral phenotype toward the reference one or vice-versa.

The intratumor heterogeneity has been recognized to characterize cancer cells impairing the efficacy of cancer treatments. We here propose an extension of constraint-based modeling approach in order to simulate metabolism of cell populations with the aim to provide a more complete characterization of these systems, especially focusing on the relationships among their components. We tested our methodology by using a toy-model and taking into account the main metabolic pathways involved in cancer metabolic rewiring. This toy-model is used as individual to construct a population model characterized by multiple interacting individuals, all having the same topology and stoichiometry, and sharing the same nutrients supply. We observed that, in our population, cancer cells cooperate with each other to reach a common objective, but without necessarily having the same metabolic traits. We also noticed that the heterogeneity emerging from the population model is due to the mismatch between the objective of the individual members and the objective of the entire population.

Mathematical modeling and computational methods facilitate the study of the functioning of biological systems and the analysis of their emergent behaviors. According to the nature of the biological system under investigation, to the available experimental data, and to the scientific question for which the model is developed, a proper mathematical formalism needs to be selected. In this work we recapitulate the results we obtained from the analysis of mechanistic models of two signal transduction pathways, outlining the relevance of the use of multi-volume models, when required, in the context of membrane systems. We also summarize some qualitative computational methods—based on the concept of communicating classes of the configurations space of membrane systems— which allow to investigate dynamical features of biological systems without the need to perform simulations. We finally discuss some general issues concerning the modeling and analysis of cellular processes.

Cell metabolism is the biochemical machinery that provides energy and building blocks to sustain life. Understanding its fine regulation is of pivotal relevance in several fields, from metabolic engineering applications to the treatment of metabolic disorders and cancer. Sophisticated computational approaches are needed to unravel the complexity of metabolism. To this aim, a plethora of methods have been developed, yet it is generally hard to identify which computational strategy is most suited for the investigation of a specific aspect of metabolism. This review provides an up-to-date description of the computational methods available for the analysis of metabolic pathways, discussing their main advantages and drawbacks. In particular, attention is devoted to the identification of the appropriate scale and level of accuracy in the reconstruction of metabolic networks, and to the inference of model structure and parameters, especially when dealing with a shortage of experimental measurements. The choice of the proper computational methods to derive in silico data is then addressed, including topological analyses, constraint-based modeling and simulation of the system dynamics. A description of some computational approaches to gain new biological knowledge or to formulate hypotheses is finally provided

Tau-leaping is a stochastic simulation algorithm that efficiently reconstructs the temporal evolution of biological systems, modeled according to the stochastic formulation of chemical kinetics. The analysis of dynamical properties of these systems in physiological and perturbed conditions usually requires the execution of a large number of simulations, leading to high computational costs. Since each simulation can be executed independently from the others, a massive parallelization of tau-leaping can bring to relevant reductions of the overall running time. The emerging field of General Purpose Graphic Processing Units (GPGPU) provides power-efficient high-performance computing at a relatively low cost. In this work we introduce cuTauLeaping, a stochastic simulator of biological systems that makes use of GPGPU computing to execute multiple parallel tau-leaping simulations, by fully exploiting the Nvidia's Fermi GPU architecture. We show how a considerable computational speedup is achieved on GPU by partitioning the execution of tau-leaping into multiple separated phases, and we describe how to avoid some implementation pitfalls related to the scarcity of memory resources on the GPU streaming multiprocessors. Our results show that cuTauLeaping largely outperforms the CPU-based tau-leaping implementation when the number of parallel simulations increases, with a break-even directly depending on the size of the biological system and on the complexity of its emergent dynamics. In particular, cuTauLeaping is exploited to investigate the probability distribution of bistable states in the Schlögl model, and to carry out a bidimensional parameter sweep analysis to study the oscillatory regimes in the Ras/cAMP/PKA pathway in S. cerevisiae

In this chapter we present an application of membrane systems to the study of intracellular diffusive processes. In particular, a class of membrane systems, called tau-DPP, is used for the modeling, simulation and analysis of bacterial chemotaxis. Two different models of this signal transduction pathway are presented. The first is a single volume model used to investigate the properties of bacterial chemotaxis and to analyze the effects of different perturbations (deletion of chemotactic proteins, addition of distinct amounts of external ligand, effect of different methylation states of the receptors) on the system dynamics. The second model represents a multivolume extension of the former, and it is exploited for the analysis of the diffusive processes that give rise to the formation of concentration gradients throughout the bacterial cytoplasm. The outcome of stochastic simulations of both models are exploited to analyze the process of synchronization of flagella, in order to evaluate the running and tumbling time intervals of bacterial cells

Constraint-based modeling is largely used in computational studies of metabolism. We propose here a novel approach that aims to identify ensembles of flux distributions that comply with one or more target phenotype(s). The methodology has been tested on a small-scale model of yeast energy metabolism. The target phenotypes describe the differential pattern of ethanol production and O2 consumption observed in "Crabtree-positive" and "Crabtree-negative" yeasts in changing environment (i.e., when the upper limit of glucose uptake is varied). The ensembles were obtained either by selection among sampled flux distributions or by means of a search heuristic (genetic algorithm). The former approach provided indication about the probability to observe a given phenotype, but the resulting ensembles could not be unambiguously partitioned into "Crabtree-positive" and "Crabtree-negative" clusters. On the contrary well-separated clusters were obtained with the latter method. The cluster analysis further allowed identification of distinct groups within each target phenotype. The method may thus prove useful in characterizing the design principles underlying metabolic plasticity arising from evolving physio-pathological or developmental constraints. © 2014 Springer Science+Business Media.

Background: The genome of living organisms is constantly exposed to several damaging agents that induce different types of DNA lesions, leading to cellular malfunctioning and onset of many diseases. To maintain genome stability, cells developed various repair and tolerance systems to counteract the effects of DNA damage. Here we focus on Post Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA).Results: We developed a novel protocol to measure the time-course ratios between mono-, di- and tri-ubiquitylated PCNA isoforms on a single western blot, which were used as the wet readout for PRR events in wild type and mutant S. cerevisiae cells exposed to acute UV radiation doses. Stochastic simulations of PCNA ubiquitylation dynamics, performed by exploiting a novel mechanistic model of PRR, well fitted the experimental data at low UV doses, but evidenced divergent behaviors at high UV doses, thus driving the design of further experiments to verify new hypothesis on the functioning of PRR. The model predicted the existence of a UV dose threshold for the proper functioning of the PRR model, and highlighted an overlapping effect of Nucleotide Excision Repair (the pathway effectively responsible to clean the genome from UV lesions) on the dynamics of PCNA ubiquitylation in different phases of the cell cycle. In addition, we showed that ubiquitin concentration can affect the rate of PCNA ubiquitylation in PRR, offering a possible explanation to the DNA damage sensitivity of yeast strains lacking deubiquitylating enzymes.Conclusions: We exploited an in vivo and in silico combinational approach to analyze for the first time in a Systems Biology context the events of PCNA ubiquitylation occurring in PRR in budding yeast cells. Our findings highlighted

The computational investigation of a biological system often requires the execution of a large number of simulations to analyze its dynamics, and to derive useful knowledge on its behavior under physiologicaland perturbed conditions. This analysis usually turns out into very high computational costs when simulations are run on central processing units (CPUs), therefore demanding a shift to the use of high performance processors. In this work we present a simulator of biological systems, called cupSODA, which exploits the higher memory bandwidth and computational capability of graphics processing units (GPUs). This software allows to execute parallel simulations of the dynamics of biological systems, by first deriving a set of ordinary differential equations from reaction-based mechanistic models defined according to the massaction kinetics, and then exploiting the numerical integration algorithm LSODA. We show that cupSODA can achieve a 112× speedup on GPUs with respect to equivalent executions of LSODA on CPUs.

The modeling of biochemical reaction networks is a fundamental but complex task in Systems Biology, which is traditionally performed exploiting human expertise and the available experimental data. Anyway, because of a general lack of knowledge on the molecular mechanisms occurring in living cells, an intense research activity focused on the development of reverse engineering methodologies is currently underway. This task is further complicated by the fact that a proper parameterization needs to be associated to the network, in order to investigate its dynamical behavior. In this work we propose a novel computational methodology for the reverse engineering of fully parameterized kinetic reaction networks, based on the combined use of two evolutionary programming techniques: Cartesian Genetic Programming (CGP) and Particle Swarm Optimization (PSO). In particular, CGP is used to infer the network topology, while PSO performs the parameter estimation task. To the purpose of applying our methodology in routine laboratory environments, we designed it to exploit a small set of experimental time series as target. We show that our methodology is able to reconstruct kinetic networks that perfectly fit with the target data

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway plays a major role in the regulation of metabolism, stress resistance and cell cycle progression. We extend here a mechanistic model of the Ras/cAMP/PKA pathway that we previously defined by describing the molecular interactions and post-translational modifications of proteins, and perform a computational analysis to investigate the dynamical behaviors of the components of this pathway, regulated by different control mechanisms. We carry out stochastic simulations to consider, in particular, the effect of the negative feedback loops on the activity of both Ira2 (a Ras-GAP) and Cdc25 (a Ras-GEF) proteins. Our results show that stable oscillatory regimes for the dynamics of cAMP can be obtained only through the activation of these feedback mechanisms, and when the amount of Cdc25 is within a specific range. In addition, we highlight that the levels of guanine nucleotides pools are able to regulate the pathway, by influencing the transition between stable steady states and oscillatory regimes.

We present a parameter estimation method, based on particle swarm optimization (PSO) and embedding the tauleaping algorithm, for the efficient estimation of reaction constants in stochastic models of biological systems, using as target a set of discrete-time measurements of molecular amounts sampled in different experimental conditions. To account for the multiplicity of data, we consider a multiswarm formulation of PSO. The whole method is developed for GPGPU architecture to reduce the computational costs. Copyright is held by the author/owner(s)

Parameter estimation (PE) of biological systems is one of the most challenging problems in Systems Biology. Here we present a PE method that integrates particle swarm optimization (PSO) to estimate the value of kinetic constants, and a stochastic simulation algorithm to reconstruct the dynamics of the system. The fitness of candidate solutions, corresponding to vectors of reaction constants, is defined as the point-to-point distance between a simulated dynamics and a set of experimental measures, carried out using discrete-time sampling and various initial conditions. A multi-swarm PSO topology with different modalities of particles migration is used to account for the different laboratory conditions in which the experimental data are usually sampled. The whole method has been specifically designed and entirely executed on the GPU to provide a reduction of computational costs.We show the effectiveness of our method and discuss its performances on an enzymatic kinetics and a prokaryotic gene expression network.

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA) on a few pivotal components of the pathway. In this article, we investigate the dynamics of the second messenger cAMP by performing stochastic simulations and parameter sweep analysis of a mechanistic model of the Ras/cAMP/PKA pathway, to determine the effects that the modulation of these feedback mechanisms has on the establishment of stable oscillatory regimes. In particular, we start by studying the role of phosphodiesterases, the enzymes that catalyze the degradation of cAMP, which represent the major negative feedback in this pathway. Then, we show the results on cAMP oscillations when perturbing the amount of protein Cdc25 coupled with the alteration of the intracellular ratio of the guanine nucleotides (GTP/GDP), which are known to regulate the switch of the GTPase Ras protein. This multi-level regulation of the amplitude and frequency of oscillations in the Ras/cAMP/PKA pathway might act as a fine tuning mechanism for the downstream targets of PKA, as also recently evidenced by some experimental investigations on the nucleocytoplasmic shuttling of the transcription factor Msn2 in yeast cells.

Systems biology is a multidisciplinary research area aimed at investigating biological systems by developing mathematical models that approach the study and the analysis of both the structure and behaviour of a biological phenomenon from a system perspective. The dynamics described by such mathematical models can be deeply affected by many parameters, and an extensive exploration of the parameters space in order to find crucial factors is most of the time prohibitive since it requires the execution of a huge number of computer simulations. Sensitivity analysis techniques can help in understanding how much the uncertainty in the model outcome is determined by the uncertainties, or by the variations, of the model input factors (components, reactions and respective parameters). In this work we exploit the European Grid Infrastructure to manage the calculations required to perform the SA on a stochastic model of bacterial chemotaxis, using an improved version of the first order screening method of Morris. According to the results achieved in our exploratory analysis, the European Grid Infrastructure is a useful solution for distributing the stochastic simulations required to carry out the SA of a stochastic model. Considering that the more intensive the computation the more scalable the infrastructure, grid computing can be a suitable technology for large scale biological models analysis

In S. cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops that can lead the second messenger cAMP to oscillatory regimes. In this paper we analyze the dynamics of cAMP by performing stochastic simulations of a mechanistic model of the Ras/cAMP/PKA pathway, to consider the effects of the deletion of phosphodiesterases (the enzymes catalyzing the degradation of cAMP, which represent the major negative feedback in this pathway) and the role of the intracellular ratio of guanine nucleotides (GTP/GDP).

In living cells, the diffusion of molecules is fundamental for the proper functioning of many intracellular processes and for their correct response to external or internal signaling. In this paper, we consider the diffusion of a pivotal protein involved in bacterial chemotaxis, and show how to model and simulate the formation of concentration gradients of this protein along the bacterial cell. For this purpose, we exploit the framework of P systems to represent the partition of the bacterial volume into separate but communicating virtual volumes, and make use of communication rules to mimic the diffusive events. We simulate this system by means of the multivolume stochastic simulation algorithm tau-DPP, and present the effects of different diffusion coefficients and of a varying number of virtual volumes over the response of the chemotactic pathway.

Chemotaxis allows bacteria to respond and adapt to the environment, by tuning tumbling and running motions due to the rotation of their flagella. We defined a model of chemotaxis and performed stochastic simulations of the dynamics of a pivotal protein, CheYp. These results allowed to compare the mean time of running, tumbling and adaptation with respect to different numbers of flagella. Our results suggest that the interplay between stochastic fluctuations of CheYp and the synchronization of flagella might represent a relevant component for the proper functionality of chemotaxis