Laboratoire P.A.S.T.E.U.R

Printer-friendly version

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Proceedings of the National Academy of Sciences, Volume 113 n°.3, January 2016, Pages 497-502

 

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a socalled fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature

Nano Letters201616 (1), pp 773–780

 

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the transcis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

Nanofibrous clinical-grade collagen scaffolds seeded with human cardiomyocytes induces cardiac remodeling in dilated cardiomyopathy

Biomaterials 80 (2016) 157-168

 

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells, this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography, the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation, the safety of the scaffold was demonstrated by echocardiography and histological assessments.

 

The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice, using cell-free scaffolds as controls, revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells, in line with the greater vascularity of the scaffold. Moreover, a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. 

Three-electrode analytical and preparative electrochemistry in micro-volume hanging droplets

Electrochem. Commun. 54 (2015) 41

 

Three-electrode micro-cells equipped with a conventional reference electrode (SCE) were easily constructed based on micro-volume droplets suspended by capillary forces to the fritted glass of the SCE bridge. Working and counter electrodes were simply inserted through the droplet surface, allowing classical electrochemistry to be readily performed in minute samples.

 

 

Light-Directed Particle Patterning by Evaporative Optical Marangoni Assembly

Nano Lett. 2015

 

Controlled particle deposition on surfaces is crucial for both exploiting collective properties of particles and their integration into devices. Most available methods depend on intrinsic properties of either the substrate or the particles to be deposited making them difficult to apply to complex, naturally occurring or industrial formulations. Here we describe a new strategy to pattern particles from an evaporating drop, regardless of inherent particle characteristics and suspension composition. We use light to generate Marangoni surface stresses resulting in flow patterns that accumulate particles at predefined positions.