Cell function profiling to assess clone stability.

In cell line development the identification of stable Chinese hamster ovary cells for production is a critical but onerous task. The stability trial focus upon high-level attributes can mask profound underlying cellular changes, leading to unstable clones mistakenly being chosen for production. The challenge is to assay underlying cell pathways and subsystems without pushing up cell line development costs. ChemStress® cell function profiling is a simple, multiwell plate-based assay that uses a panel of active chemicals to mimic known bioprocess stresses and challenge key pathways. After 3 days of static culture on the plate, functional responses are assayed, for example, titer and growth. Here this approach is used to monitor 131 clones as they change over real stability trials. A novel stability metric is defined over the data to identify stable clones that remain unperturbed across many components of cell function. This allows stability trials to look beneath the titer to identify clones that are internally more stable.

Data Management in Computational Systems Biology: Exploring Standards, Tools, Databases, and Packaging Best Practices.

Computational systems biology involves integrating heterogeneous datasets in order to generate models. These models can assist with understanding and prediction of biological phenomena. Generating datasets and integrating them into models involves a wide range of scientific expertise. As a result these datasets are often collected by one set of researchers, and exchanged with others researchers for constructing the models. For this process to run smoothly the data and models must be FAIR-findable, accessible, interoperable, and reusable. In order for data and models to be FAIR they must be structured in consistent and predictable ways, and described sufficiently for other researchers to understand them. Furthermore, these data and models must be shared with other researchers, with appropriately controlled sharing permissions, before and after publication. In this chapter we explore the different data and model standards that assist with structuring, describing, and sharing. We also highlight the popular standards and sharing databases within computational systems biology.

Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli.

Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed.

biochem4j: Integrated and extensible biochemical knowledge through graph databases.

Biologists and biochemists have at their disposal a number of excellent, publicly available data resources such as UniProt, KEGG, and NCBI Taxonomy, which catalogue biological entities. Despite the usefulness of these resources, they remain fundamentally unconnected. While links may appear between entries across these databases, users are typically only able to follow such links by manual browsing or through specialised workflows. Although many of the resources provide web-service interfaces for computational access, performing federated queries across databases remains a non-trivial but essential activity in interdisciplinary systems and synthetic biology programmes. What is needed are integrated repositories to catalogue both biological entities and-crucially-the relationships between them. Such a resource should be extensible, such that newly discovered relationships-for example, those between novel, synthetic enzymes and non-natural products-can be added over time. With the introduction of graph databases, the barrier to the rapid generation, extension and querying of such a resource has been lowered considerably. With a particular focus on metabolic engineering as an illustrative application domain, biochem4j, freely available at http://biochem4j.org, is introduced to provide an integrated, queryable database that warehouses chemical, reaction, enzyme and taxonomic data from a range of reliable resources. The biochem4j framework establishes a starting point for the flexible integration and exploitation of an ever-wider range of biological data sources, from public databases to laboratory-specific experimental datasets, for the benefit of systems biologists, biosystems engineers and the wider community of molecular biologists and biological chemists.

Recon 2.2: from reconstruction to model of human metabolism.

INTRODUCTION: The human genome-scale metabolic reconstruction details all known metabolic reactions occurring in humans, and thereby holds substantial promise for studying complex diseases and phenotypes. Capturing the whole human metabolic reconstruction is an on-going task and since the last community effort generated a consensus reconstruction, several updates have been developed. OBJECTIVES: We report a new consensus version, Recon 2.2, which integrates various alternative versions with significant additional updates. In addition to re-establishing a consensus reconstruction, further key objectives included providing more comprehensive annotation of metabolites and genes, ensuring full mass and charge balance in all reactions, and developing a model that correctly predicts ATP production on a range of carbon sources. METHODS: Recon 2.2 has been developed through a combination of manual curation and automated error checking. Specific and significant manual updates include a respecification of fatty acid metabolism, oxidative phosphorylation and a coupling of the electron transport chain to ATP synthase activity. All metabolites have definitive chemical formulae and charges specified, and these are used to ensure full mass and charge reaction balancing through an automated linear programming approach. Additionally, improved integration with transcriptomics and proteomics data has been facilitated with the updated curation of relationships between genes, proteins and reactions. RESULTS: Recon 2.2 now represents the most predictive model of human metabolism to date as demonstrated here. Extensive manual curation has increased the reconstruction size to 5324 metabolites, 7785 reactions and 1675 associated genes, which now are mapped to a single standard. The focus upon mass and charge balancing of all reactions, along with better representation of energy generation, has produced a flux model that correctly predicts ATP yield on different carbon sources. CONCLUSION: Through these updates we have achieved the most complete and best annotated consensus human metabolic reconstruction available, thereby increasing the ability of this resource to provide novel insights into normal and disease states in human. The model is freely available from the Biomodels database (http://identifiers.org/biomodels.db/MODEL1603150001).

Alcohol tax pass-through across the product and price range: do retailers treat cheap alcohol differently?

BACKGROUND AND AIMS: Effective use of alcohol duty to reduce consumption and harm depends partly on retailers passing duty increases on to consumers via price increases, also known as 'pass-through'. The aim of this analysis is to provide evidence of UK excise duty and sales tax (VAT) pass-through rates for alcohol products at different price points. SETTING: March 2008 to August 2011, United Kingdom. DESIGN AND MEASUREMENTS: Panel data quantile regression estimating the effects of three duty changes, two VAT changes and one combined duty and VAT change on UK alcohol prices, using product-level supermarket price data for 254 alcohol products available weekly. Products were analysed in four categories: beers, ciders/ready to drink (RTDs), spirits and wines. FINDINGS: Within all four categories there exists considerable heterogeneity in the level of duty pass-through for cheaper versus expensive products. Price increases for the cheapest 15% of products fall below duty rises (undershifting), while products sold above the median price are overshifted (price increases are higher than duty increases). The level of undershifting is greatest for beer [0.85 (0.79, 0.92)] and spirits [0.86 (0.83, 0.89)]. Undershifting affects approximately 67% of total beer sales and 38% of total spirits sales. CONCLUSIONS: Alcohol retailers in the United Kingdom appear to respond to increases in alcohol tax by undershifting their cheaper products (raising prices below the level of the tax increase) and overshifting their more expensive products (raising prices beyond the level of the tax increase). This is likely to impact negatively on tax policy effectiveness, because high-risk groups favour cheaper alcohol and undershifting is likely to produce smaller consumption reductions.

A community-driven global reconstruction of human metabolism.

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ∼2× more reactions and ∼1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.

The promiscuous binding of pharmaceutical drugs and their transporter-mediated uptake into cells: what we (need to) know and how we can do so.

A recent paper in this journal sought to counter evidence for the role of transport proteins in effecting drug uptake into cells, and questions that transporters can recognize drug molecules in addition to their endogenous substrates. However, there is abundant evidence that both drugs and proteins are highly promiscuous. Most proteins bind to many drugs and most drugs bind to multiple proteins (on average more than six), including transporters (mutations in these can determine resistance); most drugs are known to recognise at least one transporter. In this response, we alert readers to the relevant evidence that exists or is required. This needs to be acquired in cells that contain the relevant proteins, and we highlight an experimental system for simultaneous genome-wide assessment of carrier-mediated uptake in a eukaryotic cell (yeast).

Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast.

BACKGROUND: The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. RESULTS: To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. CONCLUSIONS: As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs.

Mining metabolites: extracting the yeast metabolome from the literature.

Text mining methods have added considerably to our capacity to extract biological knowledge from the literature. Recently the field of systems biology has begun to model and simulate metabolic networks, requiring knowledge of the set of molecules involved. While genomics and proteomics technologies are able to supply the macromolecular parts list, the metabolites are less easily assembled. Most metabolites are known and reported through the scientific literature, rather than through large-scale experimental surveys. Thus it is important to recover them from the literature. Here we present a novel tool to automatically identify metabolite names in the literature, and associate structures where possible, to define the reported yeast metabolome. With ten-fold cross validation on a manually annotated corpus, our recognition tool generates an f-score of 78.49 (precision of 83.02) and demonstrates greater suitability in identifying metabolite names than other existing recognition tools for general chemical molecules. The metabolite recognition tool has been applied to the literature covering an important model organism, the yeast Saccharomyces cerevisiae, to define its reported metabolome. By coupling to ChemSpider, a major chemical database, we have identified structures for much of the reported metabolome and, where structure identification fails, been able to suggest extensions to ChemSpider. Our manually annotated gold-standard data on 296 abstracts are available as supplementary materials. Metabolite names and, where appropriate, structures are also available as supplementary materials. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-010-0251-6) contains supplementary material, which is available to authorized users.

Pharmaceutical drug transport: the issues and the implications that it is essentially carrier-mediated only.

All cells necessarily contain tens, if not hundreds, of carriers for nutrients and intermediary metabolites, and the human genome codes for more than 1000 carriers of various kinds. Here, we illustrate using a typical literature example the widespread but erroneous nature of the assumption that the 'background' or 'passive' permeability to drugs occurs in the absence of carriers. Comparison of the rate of drug transport in natural versus artificial membranes shows discrepancies in absolute magnitudes of 100-fold or more, with the carrier-containing cells showing the greater permeability. Expression profiling data show exactly which carriers are expressed in which tissues. The recognition that drugs necessarily require carriers for uptake into cells provides many opportunities for improving the effectiveness of the drug discovery process.

Further developments towards a genome-scale metabolic model of yeast.

BACKGROUND: To date, several genome-scale network reconstructions have been used to describe the metabolism of the yeast Saccharomyces cerevisiae, each differing in scope and content. The recent community-driven reconstruction, while rigorously evidenced and well annotated, under-represented metabolite transport, lipid metabolism and other pathways, and was not amenable to constraint-based analyses because of lack of pathway connectivity. RESULTS: We have expanded the yeast network reconstruction to incorporate many new reactions from the literature and represented these in a well-annotated and standards-compliant manner. The new reconstruction comprises 1102 unique metabolic reactions involving 924 unique metabolites--significantly larger in scope than any previous reconstruction. The representation of lipid metabolism in particular has improved, with 234 out of 268 enzymes linked to lipid metabolism now present in at least one reaction. Connectivity is emphatically improved, with more than 90% of metabolites now reachable from the growth medium constituents. The present updates allow constraint-based analyses to be performed; viability predictions of single knockouts are comparable to results from in vivo experiments and to those of previous reconstructions. CONCLUSIONS: We report the development of the most complete reconstruction of yeast metabolism to date that is based upon reliable literature evidence and richly annotated according to MIRIAM standards. The reconstruction is available in the Systems Biology Markup Language (SBML) and via a publicly accessible database http://www.comp-sys-bio.org/yeastnet/.

Text mining meets workflow: linking U-Compare with Taverna.

UNLABELLED: Text mining from the biomedical literature is of increasing importance, yet it is not easy for the bioinformatics community to create and run text mining workflows due to the lack of accessibility and interoperability of the text mining resources. The U-Compare system provides a wide range of bio text mining resources in a highly interoperable workflow environment where workflows can very easily be created, executed, evaluated and visualized without coding. We have linked U-Compare to Taverna, a generic workflow system, to expose text mining functionality to the bioinformatics community. AVAILABILITY: http://u-compare.org/taverna.html, http://u-compare.org.

Integration of metabolic databases for the reconstruction of genome-scale metabolic networks.

BACKGROUND: Genome-scale metabolic reconstructions have been recognised as a valuable tool for a variety of applications ranging from metabolic engineering to evolutionary studies. However, the reconstruction of such networks remains an arduous process requiring a high level of human intervention. This process is further complicated by occurrences of missing or conflicting information and the absence of common annotation standards between different data sources. RESULTS: In this article, we report a semi-automated methodology aimed at streamlining the process of metabolic network reconstruction by enabling the integration of different genome-wide databases of metabolic reactions. We present results obtained by applying this methodology to the metabolic network of the plant Arabidopsis thaliana. A systematic comparison of compounds and reactions between two genome-wide databases allowed us to obtain a high-quality core consensus reconstruction, which was validated for stoichiometric consistency. A lower level of consensus led to a larger reconstruction, which has a lower quality standard but provides a baseline for further manual curation. CONCLUSION: This semi-automated methodology may be applied to other organisms and help to streamline the process of genome-scale network reconstruction in order to accelerate the transfer of such models to applications.

Implications of the dominant role of transporters in drug uptake by cells.

Drug entry into cells was previously believed to be via diffusion through the lipid bilayer of the cell membrane, with the contribution to uptake by transporter proteins being of only marginal importance. Now, however, drug uptake is understood to be mainly transporter-mediated. This suggests that uptake transporters may be a major determinant of idiosyncratic drug response and a site at which drug-drug interactions occur. Accurately modelling drug pharmacokinetics is a problem of Systems Biology and requires knowledge of both the transporters with which a drug interacts and where those transporters are expressed in the body. Current physiology-based pharmacokinetic models mostly attempt to model drug disposition from the biophysical properties of the drug, drug uptake by diffusion being thereby an implicit assumption. It is clear that the incorporation of transporter proteins and their drug interactions into such models will greatly improve them. We discuss methods by which tissue localisations and transporter interactions can be determined. We propose a yeast-based transporter expression system for the initial screening of drugs for their cognate transporters. Finally, the central importance of computational modelling of transporter substrate preferences by structure-activity relationships is discussed.

'Metabolite-likeness' as a criterion in the design and selection of pharmaceutical drug libraries.

Present drug screening libraries are constrained by biophysical properties that predict desirable pharmacokinetics and structural descriptors of 'drug-likeness' or 'lead-likeness'. Recent surveys, however, indicate that to enter cells most drugs require solute carriers that normally transport the naturally occurring intermediary metabolites and many drugs are likely to interact similarly. The existence of increasingly comprehensive summaries of the human metabolome allows the assessment of the concept of 'metabolite-likeness'. We compare the similarity of known drugs and library compounds to naturally occurring metabolites (endogenites) using relevant cheminformatics molecular descriptor spaces in which known drugs are more akin to such endogenites than are most library compounds.

A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology.

Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.

Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule?

It is generally thought that many drug molecules are transported across biological membranes via passive diffusion at a rate related to their lipophilicity. However, the types of biophysical forces involved in the interaction of drugs with lipid membranes are no different from those involved in their interaction with proteins, and so arguments based on lipophilicity could also be applied to drug uptake by membrane transporters or carriers. In this article, we discuss the evidence supporting the idea that rather than being an exception, carrier-mediated and active uptake of drugs may be more common than is usually assumed - including a summary of specific cases in which drugs are known to be taken up into cells via defined carriers - and consider the implications for drug discovery and development.