Petri Net computational modelling of Langerhans cell Interferon Regulatory Factor Network predicts their role in T cell activation

Langerhans cells (LCs) are able to orchestrate adaptive immune responses in the skin by interpreting the microenvironmental context in which they encounter foreign substances, but the regulatory basis for this has not been established. Utilising systems immunology approaches combining in silico modelling of a reconstructed gene regulatory network (GRN) with in vitro validation of the predictions, we sought to determine the mechanisms of regulation of immune responses in human primary LCs. The key role of Interferon regulatory factors (IRFs) as controllers of the human Langerhans cell response to epidermal cytokines was revealed by whole transcriptome analysis. Applying Boolean logic we assembled a Petri net-based model of the IRF-GRN which provides molecular pathway predictions for the induction of different transcriptional programmes in LCs. In silico simulations performed after model parameterisation with transcription factor expression values predicted that human LC activation of antigen-specific CD8 T cells would be differentially regulated by epidermal cytokine induction of specific IRF-controlled pathways. This was confirmed by in vitro measurement of IFN-g production by activated T cells. As a proof of concept, this approach shows that stochastic modelling of a specific immune networks renders transcriptome data valuable for the prediction of functional outcomes of immune responses

Identification of key regulators in glycogen utilization in E. coli based on the simulations from a hybrid functional Petri net model

Glycogen and glucose are two sugar sources available during the lag phase of E. coli, but the mechanism that regulates their utilization is still unclear

Manipulation of storage polysaccharides in microorganisms

The rising demand for arable land, to meet the competing needs of food and energy for a growing population, will soon become unmanageable. There is therefore a pressing need to increase the efficiency with which these demands are met, or find alternative ways of meeting them. In this work, E. coli transformed with a copy of its own ADP-glucose pyrophosphorylase gene (glgC), under a lac promoter, was found to synthesise large inclusion bodies when grown in media supplemented with lactose and IPTG. Analysis of these inclusion bodies suggests that they are formed of polysaccharide, giving the cell a significantly higher total sugar content than a control grown under the same conditions. The inclusion bodies were found to react strongly with iodine, turning a blackish brown colour normally associated with iodine-starch reactions. Results also indicate that the bacteria are unable to digest these inclusion bodies once they have been formed. These findings suggest the presence of long α-helices, which would both bind with iodine and prohibit enzymatic digestion. It was therefore hypothesised that the extra GlgC enzymes were allowing glucan chains to extend at a faster rate during glycogen synthesis, leading to unbranched regions that were long enough to wind themselves into helices. The subsequent introduction of a copy of the E. coli branching enzyme gene (glgB), under the same lac promoter, was therefore expected to abolish the inclusion body phenotype, by allowing the branching of the polysaccharide to keep speed with the synthesis of its linear chains. This was indeed found to be the case. However, cells transformed with both additional gene copies were also found to accumulate a significantly higher total sugar content again: more than twice that of cells transformed with glgC alone and more than seven times that of a control, when grown under conditions designed to optimise polysaccharide synthesis. These transformants were also observed to grow to higher cell densities that a control, in various growth media. The results of both these transformations could be significant in meeting the demands of our growing society. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field, providing the possibility to significantly increase yields. Lastly, the effects of Isoamylases and Granule Bound Starch Synthase, taken from two starch producing organisms – Zea mays and Ostreococcus tauri – were investigated in an E. coli host. Results were inconclusive, but suggest many avenues for continuing the work