Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells.

CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5-specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell-penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.

Electrochemical and optical investigation of dental pulp stem cell adhesion on modified porous silicon scaffolds.

Extensive use of porous silicon (PSi) for tissue engineering is due to its convenient properties as it is both nontoxic and bioresorbable. Moreover, PSi surface modification is an important step to enhance cell adhesion and proliferation. In this work, a combination of optical and electrochemical studies is performed to elaborate a suitable PSi multilayer substrate for cell culture. For this study, we modified PSi surface by silanization and antibody grafting (APTES-anti STRO1), the 12-mer specific peptide to silicon p + type coating and the peptide modified with the antibody recognition sequence. Electrochemical characterization of PSi multilayers is performed to investigate its electrical behavior, determine the optimal measuring conditions and reveal the most stable PSi surfaces. Then, the behavior of dental pulp stem cells (DPSC) was investigated on various modified PSi surfaces. An electrochemical method was applied for the first time monitoring the electrical behavior of stem cell adhesion. The cells electrochemical behavior depends on the nature of the surface coating and the peptide-anti STRO1 improved adhesion and cell spreading onto the PSi surface compared to bare surface and the one coated with the peptide. Fluorescent microscopy revealed that all surface modification methods enhance cell adhesion compared to the bare PSi surface. An increased cell number is observed on APTES-anti STRO1, peptide and peptide-anti STRO1 coated PSi. The peptide-anti STRO1 provided the best cell proliferation results suggesting the improved accessibility of the recognition fragment of the antibody anti-STRO1.