C-HiC: A High-Resolution Method for Unbiased Chromatin Conformation Capture Targeting Small Locus

International audienc

Facioscapulohumeral Muscular Dystrophy—a Tale of Heterogeneity and the Power of Clinical Assessments

International audienc

High repetition rate laser-induced prinitng of biopolymers: time-resolvestudy of multiple jet dynamics

International audienceLaser-induced forward transfer (LIFT) is a versatile, non-contact, high-resolution laser direct writing technique that offers promising solutions in various fields of application. Our work is focused on the use of this technique to create organized 2D/3D arrangements of biomaterials and living cells to create in vitro biomodels for applications in tissue engineering or regenerative medicine.LIFT is a two-part printing method using laser-matter interaction to transfer tiny amounts of material from a thin donor film to a receptor substrate, both separated by a few hundreds of micrometres. A short laser pulse induces the formation of a jet propagating perpendicularly to the donor substrate. The bio-ink previously spread as a thin film on this donor substrate is thus collected as a micrometer-sized droplet on the receiver. In order to precisely control the amount and the location of the deposited material, it is necessary to investigate carefully the jetting dynamics as a function of various parameters including the laser fluence and the rheological properties of the bioink. In this study, we used time-resolved fast imaging to investigate the hydrodynamics of the transfer of successive jets at high pulse repetition rate.The set up being currently use is composed of a high-frequency laser (12ps, 60KHz) coupled with a fast-writing scanner that allows us to print large quantities of materials in extremely short times. Successive pulses are focused on a bioink-coated donor substrate and the transfer material is imaged with a shadowgraphic technique using a delayed nanosecond flash. With this technique we can record precise moment of the dynamic and accurately decompose the ejection mechanism. We present here our investigations on the jet dynamics as a function of the rheological properties of the bioink, the irradiation fluence, the distance and time between each individual jet…Understanding and controlling these dynamics will allow us to improve the quality and reproducibility of the prints. It will also help us to estimate the ideal donor-receiver distance in order to minimize the mechanical impact of the deposition process on the cells contained in the bio-ink

Short-Pulse Lasers: A Versatile Tool in Creating Novel Nano-/Micro-Structures and Compositional Analysis for Healthcare and Wellbeing Challenges

International audienceDriven by flexibility, precision, repeatability and eco-friendliness, laser-based technologies have attracted great interest to engineer or to analyze materials in various fields including energy, environment, biology and medicine. A major advantage of laser processing relies on the ability to directly structure matter at different scales and to prepare novel materials with unique physical and chemical properties. It is also a contact-free approach that makes it possible to work in inert or reactive liquid or gaseous environment. This leads today to a unique opportunity for designing, fabricating and even analyzing novel complex bio-systems. To illustrate this potential, in this paper, we gather our recent research on four types of laser-based methods relevant for nano-/micro-scale applications. First, we present and discuss pulsed laser ablation in liquid, exploited today for synthetizing ultraclean “bare” nanoparticles attractive for medicine and tissue engineering applications. Second, we discuss robust methods for rapid surface and bulk machining (subtractive manufacturing) at different scales by laser ablation. Among them, the microsphere-assisted laser surface engineering is detailed for its appropriateness to design structured substrates with hierarchically periodic patterns at nano-/micro-scale without chemical treatments. Third, we address the laser-induced forward transfer, a technology based on direct laser printing, to transfer and assemble a multitude of materials (additive structuring), including biological moiety without alteration of functionality. Finally, the fourth method is about chemical analysis: we present the potential of laser-induced breakdown spectroscopy, providing a unique tool for contact-free and space-resolved elemental analysis of organic materials. Overall, we present and discuss the prospect and complementarity of emerging reliable laser technologies, to address challenges in materials’ preparation relevant for the development of innovative multi-scale and multi-material platforms for bio-applications

AKT Signaling Modifies the Balance between Cell Proliferation and Migration in Neural Crest Cells from Patients Affected with Bosma Arhinia and Microphthalmia Syndrome

International audienceOver the recent years, the SMCHD1 (Structural Maintenance of Chromosome flexible Hinge Domain Containing 1) chromatin-associated factor has triggered increasing interest after the identification of variants in three rare and unrelated diseases, type 2 Facio Scapulo Humeral Dystrophy (FSHD2), Bosma Arhinia and Microphthalmia Syndrome (BAMS), and the more recently isolated hypogonadotrophic hypogonadism (IHH) combined pituitary hormone deficiency (CPHD) and septo-optic dysplasia (SOD). However, it remains unclear why certain mutations lead to a specific muscle defect in FSHD while other are associated with severe congenital anomalies. To gain further insights into the specificity of SMCHD1 variants and identify pathways associated with the BAMS phenotype and related neural crest defects, we derived induced pluripotent stem cells from patients carrying a mutation in this gene. We differentiated these cells in neural crest stem cells and analyzed their transcriptome by RNA-Seq. Besides classical differential expression analyses, we analyzed our data using MOGAMUN, an algorithm allowing the extraction of active modules by integrating differential expression data with biological networks. We found that in BAMS neural crest cells, all subnetworks that are associated with differentially expressed genes converge toward a predominant role for AKT signaling in the control of the cell proliferation–migration balance. Our findings provide further insights into the distinct mechanism by which defects in neural crest migration might contribute to the craniofacial anomalies in BAMS

NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer

International audienceAlternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer

Mitochondrial function in skeletal myofibers is controlled by a TRF2‐SIRT3 axis over lifetime

International audienc

DUX4 and DUX4 downstream target genes are expressed in fetal FSHD muscles.

International audience: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult muscular dystrophies. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared muscle biopsies derived from FSHD1 fetuses and the cells derived from some of these biopsies with biopsies and cells derived from control fetuses. We mainly focus on DUX4 isoform expression because the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured DUX4 isoform expression by using qRT-PCR in fetal FSHD1 myotubes treated or not with an shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream target gene expression in myotubes and fetal or adult FSHD1 and control quadriceps biopsies. We show that both DUX4-FL isoforms are already expressed in FSHD1 myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes, which is confirmed by the level of expression of DUX4 downstream genes. We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD1 fetal quadriceps biopsies, at similar levels to those observed in adult FSHD1 quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, thus opening a new field of investigation for mechanisms leading to FSHD

SMCHD1 is involved in de novo methylation of theDUX4-encoding D4Z4 macrosatellite

International audienc