Representing dynamic biological networks with multi-scale probabilistic models.

Dynamic models analyzing gene regulation and metabolism face challenges when adapted to modeling signal transduction networks. During signal transduction, molecular reactions and mechanisms occur in different spatial and temporal frames and involve feedbacks. This impedes the straight-forward use of methods based on Boolean networks, Bayesian approaches, and differential equations. We propose a new approach, ProbRules, that combines probabilities and logical rules to represent the dynamics of a system across multiple scales. We demonstrate that ProbRules models can represent various network motifs of biological systems. As an example of a comprehensive model of signal transduction, we provide a Wnt network that shows remarkable robustness under a range of phenotypical and pathological conditions. Its simulation allows the clarification of controversially discussed molecular mechanisms of Wnt signaling by predicting wet-lab measurements. ProbRules provides an avenue in current computational modeling by enabling systems biologists to integrate vast amounts of available data on different scales.

Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection.

The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide⁻hydroquinone⁻horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out.

Concurrent epigenetic silencing of wnt/ß-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia.

BACKGROUND: The Wnt/ß-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/ß-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples. METHODS: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and ß-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. RESULTS: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/ß-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of ß-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with ß-catenin and thus demonstrating its epigenetically regulated inhibition effect. CONCLUSIONS: The results suggest an epigenetic silencing mechanism of the Wnt/ß-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

A dual array-based approach to assess the abundance and posttranslational modification state of signaling proteins.

A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of ß-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of ß-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.

Snapshots of protein dynamics and post-translational modifications in one experiment--beta-catenin and its functions.

ß-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, ß-catenin is a proto-oncogene and activating ß-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of ß-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of ß-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related ß-catenin function. The present paper describes a novel suspension bead array assay panel for ß-catenin, which requires minimal amounts of sample and is able to relatively quantify total ß-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free ß-catenin. This is the first study to combine three biochemical methods--sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay--in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different ß-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in ß-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of ß-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.

Microsphere-based co-immunoprecipitation in multiplex.

Co-immunoprecipitation (co-IP) is a prominent technique for evaluating protein-protein interactions. Currently, large quantities of protein are required to perform co-IP followed by mass spectrometric or Western blot analyses of the interacting proteins. Here catenin-cadherin complexes were employed to establish a multiplexed microsphere-based co-immunoprecipitation (micro co-IP) protocol that allows the analysis of different complexes of a given protein with various interacting proteins within a single experiment using a limited amount of sample material.