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International audience; Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.

Messenger RNA decay and processing in bacteria involve a set of various ribonucleases (RNases). In Bacillus subtilis, a newly discovered endoribonuclease called RNase Y was shown to play a central role in mRNA decay by catalyzing the initial endonucleolytic cleavage reaction. Its action releases several RNA pieces that are more sensitive to degradation by exoribonucleases. By combination of these different approaches, it was found that RNase Y seems to be organized in a oligomeric form with a ring shape. This oligomer is homogenous in microscopy, globular and structured in SAXS. The intrinsically disordered domain is not completely disordered as we can conclude with the SAXS analysis. It contains some secondary structural elements (α helix) but it has no tertiary structure as we can conclude with CD and NMR analysis respectively. SAXS analysis of the RNase Y dimeric form show that this dimeric form is structured but have a very elongated shape. In vitro activity tests on both oligomeric and dimeric form reveal that these two forms are active. Oligomer cryomicroscopy pictures collected will allow us to get a structure with a better resolution. At the moment, this oligomer seems to be composed by eight RNase Y dimer.; Le processus de maturation et de dégradation des ARNm chez les bactéries implique différentes ribonucléases (RNases). Une endoribonucléase a été découverte en 2009 chez Bacillus subtilis appelée RNase Y. Cette RNase joue un rôle central en catalysant la réaction de clivage initiatrice, un clivage endonucléolytique. Les fragments d'ARN issus du clivage par la RNase Y deviennent alors plus sensibles à la dégradation, en 5' et 3', par les exonucléases. En combinant ces différentes techniques, il s'est avéré que la RNase Y semble s'organiser sous forme d'un oligomère ayant une forme en anneau. Cet oligomère est homogène en microscopie, globulaire et structuré en SAXS. Le domaine ID lui n'est pas complètement déstructuré comme le révèle son analyse en SAXS. Il contient des éléments de structure secondaire de type hélice α et ne possède pas de structure tertiaire, comme le concluent respectivement les analyses en CD et RMN. L'analyse en SAXS de la forme dimérique de la RNase Y révèle que le dimère est structuré mais qu'il possède une forme allongée. Les tests d'activité in vitro sur la forme dimérique et oligomérique montre que ces deux formes sont activent. Les images collectées en cryo-microscopie nous permettrons d'obtenir une structure de meilleure résolution de l'oligomère, qui semble être constitué de 8 dimères de RNase Y.

Messenger RNA decay and processing in bacteria involve a set of various ribonucleases (RNases). In Bacillus subtilis, a newly discovered endoribonuclease called RNase Y was shown to play a central role in mRNA decay by catalyzing the initial endonucleolytic cleavage reaction. Its action releases several RNA pieces that are more sensitive to degradation by exoribonucleases. By combination of these different approaches, it was found that RNase Y seems to be organized in a oligomeric form with a ring shape. This oligomer is homogenous in microscopy, globular and structured in SAXS. The intrinsically disordered domain is not completely disordered as we can conclude with the SAXS analysis. It contains some secondary structural elements (α helix) but it has no tertiary structure as we can conclude with CD and NMR analysis respectively. SAXS analysis of the RNase Y dimeric form show that this dimeric form is structured but have a very elongated shape. In vitro activity tests on both oligomeric and dimeric form reveal that these two forms are active. Oligomer cryomicroscopy pictures collected will allow us to get a structure with a better resolution. At the moment, this oligomer seems to be composed by eight RNase Y dimer.; Le processus de maturation et de dégradation des ARNm chez les bactéries implique différentes ribonucléases (RNases). Une endoribonucléase a été découverte en 2009 chez Bacillus subtilis appelée RNase Y. Cette RNase joue un rôle central en catalysant la réaction de clivage initiatrice, un clivage endonucléolytique. Les fragments d'ARN issus du clivage par la RNase Y deviennent alors plus sensibles à la dégradation, en 5' et 3', par les exonucléases. En combinant ces différentes techniques, il s'est avéré que la RNase Y semble s'organiser sous forme d'un oligomère ayant une forme en anneau. Cet oligomère est homogène en microscopie, globulaire et structuré en SAXS. Le domaine ID lui n'est pas complètement déstructuré comme le révèle son analyse en SAXS. Il contient des éléments de structure secondaire de type hélice α et ne possède pas de structure tertiaire, comme le concluent respectivement les analyses en CD et RMN. L'analyse en SAXS de la forme dimérique de la RNase Y révèle que le dimère est structuré mais qu'il possède une forme allongée. Les tests d'activité in vitro sur la forme dimérique et oligomérique montre que ces deux formes sont activent. Les images collectées en cryo-microscopie nous permettrons d'obtenir une structure de meilleure résolution de l'oligomère, qui semble être constitué de 8 dimères de RNase Y.

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the “divisome”) and lateral wall growth (the “elongasome”). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors., Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.

Elevated bone marrow adiposity (BMA) is defined as an increase in the proportion of the bone marrow (BM) cavity volume occupied by adipocytes. This can be caused by an increase in the size and/or number of adipocytes. BMA increases with age in a bone-site-specific manner. This increase may be linked to certain pathophysiological situations. Osteoporosis or compromised bone quality is frequently associated with high BMA. The involvement of BM adipocytes in bone loss may be due to commitment of mesenchymal stem cells to the adipogenic pathway rather than the osteogenic pathway. However, adipocytes may also act on their microenvironment by secreting factors with harmful effects for the bone health. Here, we review evidence that in a context of energy deficit (such as anorexia nervosa (AN) and restriction rodent models) bone alterations can occur in the absence of an increase in BMA. In severe cases, bone alterations are even associated with gelatinous BM transformation. The relationship between BMA and energy deficit and the potential regulators of this adiposity in this context are also discussed. On the basis of clinical studies and preliminary results on animal model, we propose that competition between differentiation into osteoblasts and differentiation into adipocytes might trigger bone loss at least in moderate-to-severe AN and in some calorie restriction models. Finally, some of the main questions resulting from this hypothesis are discussed.

International audience; One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.