Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro.

This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.

Diffusion barriers for ADP in the cardiac cell.

The regulation of mitochondrial respiration in the intact heart may differ from that of isolated mitochondria if intracellular diffusion is restricted. Here we consider which factors may hinder diffusion in vivo and, based on computational analysis, design a reverse engineering approach to estimate the role of diffusional resistance in mitochondrial regulation from an experiment on the intact heart. Computational analysis of respiration measurements on skinned heart fibers shows that the outer mitochondrial membrane does not hinder diffusion enough to cause ADP gradients of tens of micromolars. A diffusion model further shows that the mesoscale structure of the myofibrillar space also does not hinder diffusion appreciably. However, ADP gradients are suggested by the measured activation time of oxidative phosphorylation and may be caused by diffusion restriction of other intracellular structures or the in vivo microstructure of networks of physically interacting proteins. Based on computational modeling we propose an experiment on the intact heart that allows to estimate the effective diffusion restriction between ATP producing and consuming sites in the cardiac cell.

Lightning and the Heart: Fractal Behavior in Cardiac Function.

Physical systems, from galactic clusters to diffusing molecules, often show fractal behavior. Likewise, living systems might often be well described by fractal algorithms. Such fractal descriptions in space and time imply that there is order in chaos, or put the other way around, chaotic dynamical systems in biology are more constrained and orderly than seen at first glance. The vascular network, the syncytium of cells, the processes of diffusion and transmembrane transport might be fractal features of the heart. These fractal features provide a basis which enables one to understand certain aspects of more global behavior such as atrial or ventricular fibrillation and perfusion heterogeneity. The heart might be regarded as a prototypical organ from these points of view. A particular example of the use of fractal geometry is in explaining myocardial flow heterogeneity via delivery of blood through an asymmetrical fractal branching network.