Hévéa : marqueurs protéiques de l'encoche sèche

L'apparition d'arbres "secs" dans les plantations d'hévéas et le développement de la maladie de l'encoche sèche ont suscitè des études extrêmement nombreuses. Jusqu'à présent, les causes originelles de la maladie n'ont pas été identifiées. Les facteurs génétiques intervenant au niveau moléculaire, dans les tissus producteurs, déterminent la sensibilité à la maladie de l'encoche sèche. La démarche expérimentale consiste dans la recherche, par électrophorèse bidimensionnelle, de protéines éventuellement nouvelles ou surexprimées dans le latex d'arbres malades. Les résultats présentés témoignent qu'il est possible de caractériser ces arbres par la détection de marqueurs protéiques de l'encoche sèche au niveau du latex. L'électrophorèse monodimensionnelle montre en effet des variations quantitatives au niveau d'une bande d'un poids moléculaire d'environ 22 kiloDaltons (kDa). L'intensité de la coloration de cette bande, donc la quantité en protéines, est corrélée avec la gravité de la maladi

Vacuum Casting to Manufacture a Plastic Biochip for Highly Parallel Cell Transfection

International audienceA novel polymer microarray fabrication technique is presented and applied to the realization of a biochip for highly parallelized cell transfection. The proposed microfabrication technique is derived from a macroscale rapid prototyping technique called vacuum casting. It was optimized to reduce production cost, in order to produce small series (100-10 000 chip series) of chips to meet demand in today's market of cellulomics. Microfabrication technologies and rapid prototyping technologies are combined to shape the master part, which can thus involve microsized features. The corresponding female structure is moulded in a flexible silicone material. The duplicated polymer chips are obtained by casting a thermosetting plastic under vacuum. The dimensional replication accuracy between the master part and the duplicated parts is uniform over the duplicated parts and better than 1%. Advantages of the proposed technique over existing plastic microfabrication techniques are discussed in the paper. Using this microfabrication technique, we produced a plastic biochip for highly parallelized transfection of arrays of living cells. The feasibility of parallel lipofection was demonstrated: two different plasmids encoding, respectively, eGFP and DsRED2 were inserted into HEK293T cells. The transfection was monitored through fluorescence observation after 72 h showing successful expression of both genes

Lensfree diffractive tomography for the imaging of 3D cell cultures

International audienceNew microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volume of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel ® and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm 3 of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup

Direct transfection of clonal organoids in matrigel microbeads : a promising approach toward organoid-based genetic screens

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications

A microfluidic platform integrating functional vascularized organoids-on-chip

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics

Natural Single-Nucleosome Epi-Polymorphisms in Yeast

Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions