The development of high quality seals for silicon patch-clamp chips.

International audiencePlanar patch-clamp is a two-dimensional variation of traditional patch-clamp. By contrast to classical glass micropipette, the seal quality of silicon patch-clamp chips (i.e. seal resistance and seal success rate) have remained poor due to the planar geometry and the nature of the substrate and thus partially obliterate the advantages related to planar patch-clamp. The characterization of physical parameters involved in seal formation is thus of major interest. In this paper, we demonstrate that the physical characterization of surfaces by a set of techniques (Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), surface energy (polar and dispersive contributions), drop angles, impedance spectroscopy, combined with a statistical design of experiments (DOE)) allowed us discriminating chips that provide relevant performances for planar patch-clamp analysis. Analyses of seal quality demonstrate that dispersive interactions and micropore size are the most crucial physical parameters of chip surfaces, by contrast to surface roughness and dielectric membrane thickness. This multi-scale study combined with electrophysiological validation of chips on a diverse set of cell-types expressing various ion channels (IRK1, hERG and hNa(v)1.5 channels) unveiled a suitable patch-clamp chip candidate. This original approach may inspire novel strategies for selecting appropriate surface parameters dedicated to biochips

The miR-17 family links p63 protein to MAPK signaling to promote the onset of human keratinocyte differentiation.

The p63 protein plays a key role in regulating human keratinocyte proliferation and differentiation. Although some p63-regulating microRNAs (miRNAs) have been identified in the control of epidermal homeostasis, little is known about miRNAs acting downstream of p63. In this paper, we characterized multiple p63-regulated miRNAs (miR-17, miR-20b, miR-30a, miR-106a, miR-143 and miR-455-3p) and elucidated their roles in the onset of keratinocyte differentiation. We identified RB, p21 and multiple MAPKs as targets of these p63-controlled miRNAs. Upon inhibition of most of these miRNAs, we observed defects in commitment to differentiation that could be reversed by siRNA-mediated silencing of their targets. Furthermore, knockdown of MAPK8 and MAPK9 efficiently restored expression of the early differentiation markers keratin 1 and keratin 10 in p63-silenced primary human keratinocytes. These results highlight new mechanistic roles of multiple miRNAs, particularly the miR-17 family (miR-17, miR-20b and miR-106a), as regulatory intermediates for coordinating p63 with MAPK signaling in the commitment of human mature keratinocytes to early differentiation

Table of the dielectric parameters for three human cell lines.

<p>The cytoplasm conductivity σ₃ and membrane capacitance <i>C_mem</i> are calculated from experimental fit to the competitive model.</p

Response of HEK cells to dielectrophoresis for increasing medium conductivities.

<p>nDEP and pDEP are applied at <i>f</i>  =  1 kHz and <i>f</i>  =  200 kHz, respectively. The arrow represents cell motion during 5 frames (300 ms), the picture being the last image. DEP is stronger at low conductivities compared to EHD forces so cells experience larger displacement at higher velocities at low conductivities.</p

Rotation study of three human cell lines.

<p><b>(a1)</b> Time-lapse sequence images of the rotation of HEK cells in the z-axis, in presence of 1 µm polystyrene colloid (highlighted in blue circles). Particles were added to observe medium stream lines. The red circle pinpoints a visible organelle. Rotation is studied at σ_m  =  2.10⁻² S/m when varying <b>(a2)</b> magnitude of the electric field at <i>f</i>  =  45 kHz or <b>(a3)</b> frequency at magnitude 0.065 V/µm (V = 10 Vp-p). The dashed line plots the values of |Re[CMF(ω)]| at the same frequencies, bringing out the relation between DEP effect and ETE. Rotation studies of <b>(b)</b> of JURKAT cells and <b>(c)</b> PC3 cells (electric field magnitude is 0.089 V/µm (V = 4Vp-p) and σ_m  =  2 10⁻² S/m.). The inset on the lower part of the graphs shows the number of cells used for each mean value.</p

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

International audienceA simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs

Single shell model of a mamalian cell with dielectric parameters annoted.

<p>Single shell model of a mamalian cell with dielectric parameters annoted.</p

Summary of cells behaviors at (a) σ_m = 2.10⁻⁴ S/m and (b) σ_m = 2.10⁻² S/m.

<p>Photographs and schemes illustrate the cell motion for typical frequencies and magnitudes with corresponding graphs of the DEP (U_DEP, green line) and EHD (U_EHD, in red) velocities. The velocities were calculated according to the theoretical model presented in the first paragraph and position of the field was taken for x = 1 µm. The boxed text refers to the related paragraph.</p

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

International audienceAbstract Background and Aims Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. Methods We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. Results siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. Conclusions The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases