Franco B. du Preez

From steady-state to synchronized yeast glycolytic oscillations I: Model construction

An existing detailed kinetic model for the steady‐state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit‐cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter‐and intra‐cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J279, 2823–2836], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re‐use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative.

Glutathione metabolism modeling: A mechanism for liver drug-robustness and a new biomarker strategy

BACKGROUND Glutathione metabolism can determine an individuals ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). METHODS Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. RESULTS Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. CONCLUSION A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. GENERAL SIGNIFICANCE Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.

From steady-state to synchronized yeast glycolytic oscillations II: model validation.

In an accompanying paper [du Preez et al., (2012) FEBS J279, 2810–2822], we adapt an existing kinetic model for steady‐state yeast glycolysis to simulate limit‐cycle oscillations. Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out‐of‐phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose‐ and acetaldehyde‐driven forced oscillations, glucose and acetaldehyde quenching, and cell‐free extract oscillations (including complex oscillations and mixed‐mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets.

A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid concentrations with increased dosage of paracetamol. Experimental studies have thus far failed to resolve these paradoxes and the robustness of how these proposed biomarkers correlate with liver glutathione levels has been questioned. To better understand how these biomarkers behave in the glutathione system a kinetic model of this pathway was made. By using metabolic control analysis and by simulating biomarker levels under a variety of conditions, we found that 5-oxoproline and ophthalmic acid concentrations may not only depend on the glutathione but also on the methionine status of the cell. We show that neither of the two potential biomarkers are reliable on their own since they need additional information about the methionine status of the system to relate them uniquely to intracellular glutathione concentration. However, when both biomarkers are measured simultaneously a direct inference of the glutathione concentration can be made, irrespective of the methionine concentration in the system.

RightField: scientific knowledge acquisition by stealth through ontology-enabled spreadsheets

RightField is a Java application that provides a mechanism for embedding ontology annotation support for scientific data in Microsoft Excel or Open Office spreadsheets. The result is semantic annotation by stealth, with an annotation process that is less error-prone, more efficient, and more consistent with community standards. By automatically generating RDF statements for each cell a rich, Linked Data querying environment allows scientists to search their data and other Linked Data resources interchangeably, and caters for queries across heterogeneous spreadsheets. RightField has been developed for Systems Biologists but has since adopted more widely. It is open source (BSD license) and freely available from http://www.rightfield.org.uk.

Stealthy annotation of experimental biology by spreadsheets

The increase in volume and complexity of biological data has led to increased requirements to reuse that data. Consistent and accurate metadata is essential for this task, creating new challenges in semantic data annotation and in the constriction of terminologies and ontologies used for annotation. The BioSharing community are developing standards and terminologies for annotation, which have been adopted across bioinformatics, but the real challenge is to make these standards accessible to laboratory scientists. Widespread adoption requires the provision of tools to assist scientists whilst reducing the complexities of working with semantics. This paper describes unobtrusive ‘stealthy’ methods for collecting standards compliant, semantically annotated data and for contributing to ontologies used for those annotations. Spreadsheets are ubiquitous in laboratory data management. Our spreadsheet‐based RightField tool enables scientists to structure information and select ontology terms for annotation within spreadsheets, producing high quality, consistent data without changing common working practices. Furthermore, our Populous spreadsheet tool proves effective for gathering domain knowledge in the form of Web Ontology Language (OWL) ontologies. Such a corpus of structured and semantically enriched knowledge can be extracted in Resource Description Framework (RDF), providing further means for searching across the content and contributing to Open Linked Data (http://linkeddata.org/). Copyright