Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

BACKGROUND: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. RESULTS: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. CONCLUSIONS: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses

Intelligent Mobility Systems:Some Socio-technical Challenges and Opportunities

Analysis of socio-technical challenges and opportunities around contemporary mobilities suggests new interpretations and visions for intelligent transport systems. Multiple forms of intelligence are required (but not easily compatible), transport is too narrow a term, and innovation results in new sociotechnical systems. An exploration of cumulative, collective and collaborative aspects of mobility systems, allows us to sketch challenges and opportunities in relation to practices of collaboration, communication and coordination, literacies for creativity, comfort and control, citizenship and (lack of) a sense of crisis, concluding with a discussion of methodological implications