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International audience; Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.

The flesh of the Pinctada radiata pearl oyster from coastal Tunisia is considered as a high source of n-3 and n-6 and its shell nacre layer is a promising osteogenic biomaterial. Fatty acid (FA) analysis showed that the major components found in total FA (TFA) were 14:0, 16:0, and 18:0 saturated FA (SFA); 16:1, 18:1, and 20:1 monoenoic FA; 20:4n-6 (ARA), 22:5n-3 (DPA). Characteristically high levels of 20:5n-3 (EPA) and 22:6n-3 (DHA) (6.53-89.75 mg/100 g TFA) polyunsaturated FA (PUFA) were found, respectively, in the TFA of nacre and flesh. Evaluated the effects in vitro of lipids extracted from nacre (Ln) and from flesh (Lc) of P. radiata on growth and the differentiation of osteoblasts. Cytotoxicity tests (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide [MTT] and lactic acid dehydrogenase c [LDH]) demonstrated that both extracts are nontoxic. Alizarin Red staining was used in an osteoblast differentiation model using the osteoblast MC3T3-E1 cell line. It showed that the FA of both extracts induced osteoblast differentiation leading to mineralization. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significantly higher expression of osteocalcin (Bglap) and runt-related transcription (Runx2) in MC3T3-E1 cells in the presence of Ln. No difference of osteopontin (Spp1) and Collagen type I (Col1a1) genes compared to the control was observed. In conclusion, these results supported, obtained from our in vitro experimental model used, the interest/potential of lipids extracted from nacre and P. radiata flesh to stimulate bone formation.

During the past two decades, with a huge and rapidly increasing clinical need for bone regeneration and repair, bone substitutes are more and more seen as a potential solution. Major innovation efforts are being made to develop such substitutes, some having advanced even to clinical practice. It is now time to turn to natural biomaterials. Nacre, or mother-of-pearl, is an organic matrix-calcium carbonate coupled shell structure produced by molluscs. In vivo and in vitro studies have revealed that nacre is osteoinductive, osteoconductive, biocompatible, and biodegradable. With many other outstanding qualities, nacre represents a natural and multi-use biomaterial as a bone graft substitute. This review aims at summarising the current needs in orthopaedic clinics and the challenges for the development of bone substitutes; most of all, we systematically review the physiological characteristics and biological evidence of nacre's effects centred on osteogenesis, and finally we put forward the potential use of nacre as a bone graft substitute. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 662-671, 2017.

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl2 was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl2 differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.

International audience; DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.

Eukaryotic chromosomes undergo movements that are involved in the regulation of functional processes such as DNA repair. To better understand the origin of these movements, we used fluorescence microscopy, image analysis and chromosome conformation capture to quantify the actin contribution to chromosome movements and interactions in budding yeast. We show that both the cytoskeletal and nuclear actin drive local chromosome movements, independently of Csm4, a putative LINC protein. Inhibition of actin polymerization reduces subtelomere dynamics, resulting in more confined territories and enrichment in subtelomeric contacts. Artificial tethering of actin to nuclear pores increased both nuclear pore complex (NPC) and subtelomere motion. Chromosome loci that were positioned away from telomeres exhibited reduced motion in the presence of an actin polymerization inhibitor but were unaffected by the lack of Csm4. We further show that actin was required for locus mobility that was induced by targeting the chromatin-remodeling protein Ino80. Correlated with this, DNA repair by homologous recombination was less efficient. Overall, interphase chromosome dynamics are modulated by the additive effects of cytoskeletal actin through forces mediated by the nuclear envelope and nuclear actin, probably through the function of actin in chromatin-remodeling complexes.