2017
- Two-Photon Photosensitizer Polymer Conjugates for Combined Cancer Cell Death Induction and Two-Photon Fluorescence Imaging: Structure/Photodynamic Therapy Efficiency Relationship
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Cepraga, C; Marotte, S; Ben Daoud, E; Favier, A; Lanoe, PH; Monnereau, C; Baldeck, P; Andraud, C; Marvel, J; Charreyre, MT; Leverrier, Y
- BIOMACROMOLECULES
- Acknowledged platforms: PLATIM, Cytometry
Abstract :
One of the challenges of photodynamic therapy is to increase the penetration depth of light irradiation in the tumor tissues. Although two-photon excitation strategies have been developed, the two-photon absorption cross sections of clinically used photosensitizers are generally low (below 300 GM). Besides, photosensitizers with high cross section values are often non-water-soluble. In this research work, a whole family of photosensitizer-polymer conjugates was synthesized via the covalent binding of a photosensitizer with a relatively high cross section along a biocompatible copolymer chain. The resulting photosensitizer-polymer conjugates were water-soluble and could be imaged in cellulo by two-photon microscopy thanks to their high two-photon absorption cross sections (up to 2600 GM in water, in the NIR range). In order to explore the structure/photodynamic activity relationship of such macromolecular photosensitizers, the influence of the polymer size, photosensitizer density, and presence of charges along the polymer backbone was investigated (neutral, anionic, cationic, and zwitterionic conjugates were compared). The macromolecular photosensitizers were not cytotoxic in the absence of light irradiation. Their kinetics of cellular uptake in the B16-F10 melanoma cell line were followed by flow cytometry over 24 h. The efficiency of cell death upon photoactivation was found to be highly correlated to the cellular uptake in turn correlated to the global charge of the macromolecular photosensitizer which appeared as the determining structural parameter.
DEC 2017
DOI : 10.1021/acs.biomac.7b01090
Pubmed : 29020442
- D-Alanylation of teichoic acids contributes to Lactobacillus plantarum-mediated Drosophila growth during chronic undernutrition
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Matos, RC; Schwarzer, M; Gervais, H; Courtin, P; Joncour, P; Gillet, B; Ma, DL; Bulteau, AL; Martino, ME; Hughes, S; Chapot-Chartier, MP; Leulier, F
- NATURE MICROBIOLOGY
- Acknowledged platforms: PLATIM, Arthro-Tools
Abstract :
. The microbial environment influences animal physiology. However, the underlying molecular mechanisms of such functional interactions are largely undefined. Previously, we showed that during chronic undernutrition, strains of Lactobacillus plantarum, a major commensal partner of Drosophila, promote host juvenile growth and maturation partly through enhanced expression of intestinal peptidases. By screening a transposon insertion library of Lactobacillus plantarum in gnotobiotic Drosophila larvae, we identify a bacterial cell-wall-modifying machinery encoded by the pbpX2-dlt operon that is critical to enhance host digestive capabilities and promote animal growth and maturation. Deletion of this operon leads to bacterial cell wall alteration with a complete loss of D-alanylation of teichoic acids. We show that L. plantarum cell walls bearing D-alanylated teichoic acids are directly sensed by Drosophila enterocytes to ensure optimal intestinal peptidase expression and activity, juvenile growth and maturation during chronic undernutrition. We thus conclude that besides peptidoglycan, teichoic acid modifications participate in the host-commensal bacteria molecular dialogue occurring in the intestine.
DEC 2017
DOI : 10.1038/s41564-017-0038-x
Pubmed : 28993620
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Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages
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Latroche, C; Weiss-Gayet, M; Muller, L; Gitiaux, C; Leblanc, P; Liot, S; Ben-Larbi, S; Abou-Khalil, R; Verger, N; Bardot, P; Magnan, M; Chretien, F; Mounier, R; Germain, S; Chazaud, B
- STEM CELL REPORTS
- Acknowledged platform: Cytometry
Abstract :
In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling.
DEC 2017
DOI: 10.1016/j.stemcr.2017.10.027
Pubmed : 29198825
- Molecular dissection of protein-protein interactions between integrin alpha 5 beta 1 and the Helicobacter pylori Cag type IV secretion system
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Koelblen, T; Berge, C; Cherrier, MV; Brillet, K; Jimenez-Soto, L; Ballut, L; Takagi, J; Montserret, R; Rousselle, P; Fischer, W; Haas, R; Fronzes, R; Terradot, L
- FEBS JOURNAL
- Acknowledged platform: PSF
Abstract :
The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagY(RRII)), CagY C-terminal domain (CagY(B10)) and integrin 51 ectodomain (51(E)) or headpiece domain (51(HP)). We found that CagI, CagA, CagL and CagY(B10) but not CagY(RRII) were able to interact with 51(E) with affinities similar to the one observed for 51(E) interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagY(B10) affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagY(B10) interacted with the 51(HP). Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagY(B10), and CagA and CagI. Altogether, our data map the network of interactions between host-cell 51 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection.
DEC 2017
DOI : 10.1111/febs.14299
Pubmed : 29055076
- Potential Role of OCT4 in Leukemogenesis
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Picot, T; Kesr, S; Wu, Y; Aanei, CM; Flandrin-Gresta, P; Tondeur, S; Tavernier, E; Wattel, E; Guyotat, D; Campos, L
- STEM CELLS AND DEVELOPMENT
- Acknowledged platform: Vectorology
Abstract :
Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.
NOV 2017
DOI : 10.1089/scd.2017.0134
Pubmed : 28911263
- The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs
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Panthu, B; Terrier, O; Carron, C; Traversier, A; Corbin, A; Balvay, L; Lina, B; Rosa-Calatrava, M; Ohlmann, T
-
JOURNAL OF MOLECULAR BIOLOGY
-
Acknowledged platform: PSF
Abstract :
The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs.
OCT 2017
DOI : 10.1016/j.jmb.2017.04.007
Pubmed : 28433538
- How aging impacts skin biomechanics: a multiscale study in mice
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Lynch, B; Bonod-Bidaud, C; Ducourthial, G; Affagard, JS; Bancelin, S; Psilodimitrakopoulos, S; Ruggiero, F; Allain, JM; Schanne-Klein, MC
- SCIENTIFIC REPORTS
- Acknowledged platform: PBES
Abstract :
Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice. The simultaneous variations of a full set of mechanical and microstructural parameters are analyzed in the framework of a multiscale mechanical interpretation. They show consistent results for wild-type mice as well as for genetically-modified mice with modified collagen V synthesis. We mainly observe an increase of the tangent modulus and a lengthening of the heel region in old murine skin from all strains, which is attributed to two different origins that may act together: (i) increased cross-linking of collagen fibers and (ii) loss of water due to proteoglycans deterioration, which impedes inner sliding within these fibers. In contrast, the microstructure reorganization upon stretching shows no age-related difference, which can be attributed to opposite effects of the decrease of collagen content and of the increase of collagen cross-linking in old mice.
OCT 2017
DOI : 10.1038/s41598-017-13150-4
Pubmed : 29061975
- Expanding the Kinome World: A New Protein Kinase Family eWidely Conserved in Bacteria
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Nguyen, HA; El Khoury, T; Guiral, S; Laaberki, MH; Candusso, MP; Galisson, F; Foucher, AE; Kesraoui, S; Ballut, L; Vallet, S; Orelle, C; Zucchini, L; Martine, J; Page, A; Attieh, J; Aghajari, N; Grangeasse, C; Jault,
- JOURNAL OF MOLECULAR BIOLOGY
- Collaborative platform: PSF; Acknowledged platform: PSF
Abstract :
Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0 angstrom) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a Delta ydiB mutant was more sensitive to paraquat treatment than the wild type, with similar to 13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis.
OCT 2017
DOI : 10.1016/j.jmb.2017.08.016
Pubmed : 28890133
- Microenvironment-Driven Shift of Cohesion/Detachment Balance within Tumors Induces a Switch toward Metastasis in Neuroblastoma
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Delloye-Bourgeois, C; Bertin, L; Thoinet, K; Jarrosson, L; Kindbeiter, K; Buffet, T; Tauszig-Delamasure, S; Bozon, M; Marabelle, A; Combaret, V; Bergeron, C; Derrington, E; Castellani, V
-
CANCER CELL
-
Acknowledged platform: Vectorology
Abstract :
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.
OCT 2017
DOI : 10.1016/j.ccell.2017.09.006
Pubmed : 29017055
- Gradient reconstitution of membrane proteins for solid-state NMR studies
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Lacabanne, D; Lends, A; Danis, C; Kunert, B; Fogeron, ML; Jirasko, V; Chuilon, C; Lecoq, L; Orelle, C; Chaptal, V; Falson, P; Jault, JM; Meier, BH; Bockmann, A
- JOURNAL OF BIOMOLECULAR NMR
- Acknowledged platform: PSF
Abstract :
We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.
OCT 2017
DOI : 10.1007/s10858-017-0135-4
Pubmed : 28900789
- Dual "mAb" HER family blockade in head and neck cancer human cell lines combined with photon therapy
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Guy, JB; Mery, B; Ollier, E; Espenel, S; Vallard, A; Wozny, AS; Simonet, S; Lauret, A; Battiston-Montagne, P; Ardail, D; Alphonse, G; Rancoule, C; Rodriguez-Lafrasse, C; Magne, N
- SCIENTIFIC REPORTS
- Acknowledged platform: Cytometry
Abstract :
Head and neck cancer stem cells (CSCs) are highly resistant to treatment. When EGFR is overexpressed in head and neck squamous cell carcinoma (HNSCC), HER2 and HER3 are also expressed. The aim of the present study was to investigate the effect of HER1/2/3 blockade through a combination of cetuximab and pertuzumab, with or without photon irradiation, on the proliferation and migration/invasion capabilities of an HNSCC chemo-and radioresistant human cell line (SQ20B) and its corresponding stem cell subpopulation. Cell proliferation, migration and invasion were studied after treatment with cetuximab(+/-) pertuzumab(+/-) 10 Gy photon irradiation. EGFR, phospho-EGFR, HER2 and HER3 protein expression levels were studied. Activation or inhibition of the RAS/MAPK and AKT-mTOR downstream signalling cascades was investigated through phospho-AKT and phosphoMEK1/2 expression. Cetuximab strongly inhibited SQ20B and FaDu cell proliferation, migration and invasion, whereas it had little effect on SQ20B-CSCs. Cetuximab-pertuzumab combined with radiation significantly inhibited SQ20B and FaDu cell and SQ20B-CSC proliferation, migration and invasion. Cetuximab-pertuzumab with 10 Gy photon irradiation switched off both phospho-AKT and phosphoMEK1/2 expression in the three populations. The triple therapy is therefore thought to inhibit SQ20B cells, SQ20B-CSCs and FaDu cells through an AKT-mTOR and Ras-MAPK downstream signalling blockade.
SEP 2017
DOI : 10.1038/s41598-017-12367-7
Pubmed : 28939847
- The C. elegans SET-2/SET1 histone H3 Lys4 (H3K4) methyltransferase preserves genome stability in the germline
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Herbette, M; Mercier, MG; Michal, F; Cluet, D; Burny, C; Yvert, G; Robert, VJ; Palladino, F
- DNA REPAIR
- Acknowledged platform: PLATIM
Abstract :
Maintaining the integrity of genetic information across generations is essential for both cell survival and reproduction, and requires the timely repair of DNA damage. Histone-modifying enzymes play a central role in the DNA repair process through the deposition and removal of post-translational modifications on the histone tails. Specific histone modification act in the DNA repair process through the recruitment of proteins and complexes with specific enzymatic activities, or by altering the chromatin state at the site of DNA lesions. The conserved SET1/MLL family of histone methyltransferases (HMT) catalyzes methylation of histone H3 on Lysine 4 (H3K4), a histone modification universally associated with actively transcribed genes. Studies have focused on the role of SET1/MLL proteins in epigenetic regulation of gene expression. Much less is known on their role in the DNA repair process in a developmental context. Here we show that SET-2, the Caenorhabditis elegans orthologue of SET1, is required to preserve germline genome integrity over subsequent generations. Animals lacking the SET-2 catalytic subunit show a transgenerational increase in sensitivity to DNA damage-inducing agents that is accompanied by a defect in double-strand break (DSB) repair and chromosome fragmentation. These defects are not due to a failure to activate the DNA damage response (DDR) that allows detection, signaling and repair of DNA lesions, because cell cycle arrest and apoptosis, key components of this pathway, are efficiently induced in set-2 mutant animal. Rather, our results suggest that SET-2 plays a role in the transgenerational maintenance of genome stability by acting in DNA repair downstream of DDR signaling.
SEP 2017
DOI : 10.1016/j.dnarep.2017.07.007
Pubmed : 28779964
- SIRT1 regulates macrophage self-renewal
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Imperatore, F; Maurizio, J; Aguilar, SV; Busch, CJ; Favret, J; Kowenz-Leutz, E; Cathou, W; Gentek, R; Perrin, P; Leutz, A; Berruyer, C; Sieweke, MH
- EMBO JOURNAL
- Acknowledged platform: Vectorology
Abstract :
Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo. Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.
AUG 2017
DOI : 10.15252/embj.201695737
Pubmed : 28701484
- Methylcytosine dioxygenase TET3 interacts with thyroid hormone nuclear receptors and stabilizes their association to chromatin
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Guan, WY; Guyot, R; Samarut, J; Flamant, F; Wong, JM; Gauthier, KC
-
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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Acknowledged platform: Cytometry
Abstract :
Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily that act as ligand-dependent transcription factors. Here we identified the ten-eleven translocation protein 3 (TET3) as a TR interacting protein increasing cell sensitivity to T3. The interaction between TET3 and TRs is independent of TET3 catalytic activity and specifically allows the stabilization of TRs on chromatin. We provide evidence that TET3 is required for TR stability, efficient binding of target genes, and transcriptional activation. Interestingly, the differential ability of different TR alpha 1 mutants to interact with TET3 might explain their differential dominant activity in patients carrying TR germline mutations. So this study evidences a mode of action for TET3 as a nonclassical coregulator of TRs, modulating its stability and access to chromatin, rather than its intrinsic transcriptional activity. This regulatory function might be more general toward nuclear receptors. Indeed, TET3 interacts with different members of the superfamily and also enhances their association to chromatin.
AUG 2017
DOI : 10.1073/pnas.1702192114
Pubmed : 28716910
- Topical ivermectin improves allergic skin inflammation
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Ventre, E; Rozieres, A; Lenief, V; Albert, F; Rossio, P; Laoubi, L; Dombrowicz, D; Staels, B; Ulmann, L; Julia, V; Vial, E; Jomard, A; Hacini-Rachinel, F; Nicolas, JF; Vocanson, M
-
ALLERGY
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Acknowledged platform: Cytometry
Abstract :
Background: Ivermectin (IVM) is widely used in both human and veterinary medicine to treat parasitic infections. Recent reports have suggested that IVM could also have anti-inflammatory properties. Methods: Here, we investigated the activity of IVM in a murine model of atopic dermatitis (AD) induced by repeated exposure to the allergen Dermatophagoides farinae, and in standard cellular immunological assays. Results: Our results show that topical IVM improved allergic skin inflammation by reducing the priming and activation of allergen-specific T cells, as well as the production of inflammatory cytokines. While IVM had no major impact on the functions of dendritic cells in vivo and in vitro, IVM impaired T-cell activation, proliferation, and cytokine production following polyclonal and antigen-specific stimulation. Conclusion: Altogether, our results show that IVM is endowed with topical anti-inflammatory properties that could have important applications for the treatment of T-cell-mediated skin inflammatory diseases.
AUG 2017
DOI : 10.1111/all.13118
Pubmed : 28052336
- A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors
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Imbert, PRC; Louche, A; Luizet, JB; Grandjean, T; Bigot, S; Wood, TE; Gagne, S; Blanco, A; Wunderley, L; Terradot, L; Woodman, P; Garvis, S; Filloux, A; Guery, B; Salcedo, SP
- EMBO JOURNAL
- Acknowledged platform: PSF
Abstract :
Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain-containing protein (PumA) of the multi-drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF-kappa B, a property transferable to non-PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll-like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin-associated protein 1 (UBAP1), a component of the endosomal-sorting complex required for transport I (ESCRT-I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.
JUL 2017
DOI : 10.15252/embj.201695343
Pubmed : 28483816
- AMPK alpha 1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis
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Theret, M; Gsaier, L; Schaffer, B; Juban, G; Ben Larbi, S; Weiss-Gayet, M; Bultot, L; Collodet, C; Foretz, M; Desplanches, D; Sanz, P; Zang, ZZ; Yang, L; Vial, G; Viollet, B; Sakamoto, K; Brunet, A; Chazaud, B; Mounier, R
- EMBO JOURNAL
- Acknowledged platform: Cytometry
Abstract :
Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPK alpha 1(-/-) MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPK alpha 1(-/-) MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPK alpha 1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPK alpha 1(-/-) phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPK alpha 1(-/-) MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPK alpha 1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.
JUL 2017
DOI : 10.15252/embj.201695273
Pubmed : 2815121
- Infrasonic and Ultrasonic Hearing Evolved after the Emergence of Modern Whales
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Mourlam, MJ; Orliac, MJ
- CURRENT BIOLOGY
- Acknowledged platform: ImmOs
Abstract :
Mysticeti (baleen whales) and Odontoceti (toothed whales) today greatly differ in their hearing abilities: Mysticeti are presumed to be sensitive to infrasonic noises [1-3], whereas Odontoceti are sensitive to ultrasonic sounds [4-6]. Two competing hypotheses exist regarding the attainment of hearing abilities in modern whales: ancestral low-frequency sensitivity [7-13] or ancestral high-frequency sensitivity [14, 15]. The significance of these evolutionary scenarios is limited by the undersampling of both early-diverging cetaceans (archaeocetes) and terrestrial hoofed relatives of cetaceans (non-cetacean artiodactyls). Here, we document for the first time the bony labyrinth, the hollow cavity housing the hearing organ, of two species of protocetid whales from Lutetian deposits (ca. 46-43 Ma) of Kpogame, Togo. These archaeocete cetaceans, which are transitional between terrestrial and aquatic forms, prove to be a key for determining the hearing abilities of early whales. We propose a new evolutionary picture for the early stages of this history, based on qualitative and quantitative studies of the cochlear morphology of an unparalleled sample of extant and extinct land artiodactyls and cetaceans. Contrary to the hypothesis that archaeocetes have been more sensitive to high-frequency sounds than their terrestrial ancestors [15], we demonstrate that early cetaceans presented a cochlear functional pattern close to that of their terrestrial relatives, and that specialization for infrasonic or ultrasonic hearing in Mysticeti or Odontoceti, respectively, instead only occurred in fully aquatic whales, after the emergence of Neoceti (Mysticeti+Odontoceti).
JUN 2017
DOI : 10.1016/j.cub.2017.04.061
Pubmed : 28602653
- Differential pattern of HIF-1 alpha expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect
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Wozny, AS; Lauret, A; Battiston-Montagne, P; Guy, JB; Beuve, M; Cunha, M; Saintigny, Y; Blond, E; Magnel', N; LaIlel, P; Ardail, D; Alphonse, G; Rodriguez-Lafrasse, C
- BRITISH JOURNAL OF CANCER
- Acknowledged platform: Cytometry
Abstract :
Background: Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. Methods: Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1 alpha. Results: Under hypoxia, compared to non-CSCs, HIF-1 alpha is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1 alpha expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1 alpha expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1 alpha. Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1 alpha inhibition. Conclusions: Hypoxia-inducible factor-1 alpha plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1 alpha targeting an attractive therapeutic challenge.
MAY 2017
DOI : 10.1038/bjc.2017.100
Pubmed : 28407653
- Distinct Contributions of Autophagy Receptors in Measles Virus Replication
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Petkova, DS; Verlhac, P; Rozieres, A; Baguet, J; Claviere, M; Kretz-Remy, C; Mahieux, R; Viret, C; Faure, M
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VIRUSES-BASEL
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Acknowledged platforms: Cytometry, PLATIM
Abstract :
Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO2 and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.
MAY 2017
DOI : 10.3390/v9050123
Pubmed : 28531150
- Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes.
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Doumane, M; Lionnet, C; Bayle, V; Jaillais, Y; Caillaud, MC
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BIO PROTOC
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Collaborative platform: PLATIM
Abstract :
Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amendable to automatically track non-plant structures with an apical growth.
APR 2017
DOI : 10.21769/BioProtoc.2245.
Pubmed : 28459086
- Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny
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Crauste, F; Mafille, J; Boucinha, L; Djebali, S; Gandrillon, O; Marvel, J; Arpin, C
- CELL SYSTEMS
- Acknowledged platforms: Cytometry, PBES
Abstract :
Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive/early effector/late effector/memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.
MAR 2017
DOI : 10.1016/j.cels.2017.01.014
Pubmed : 28237797
- Neuronal expression of a thyroid hormone receptor alpha mutation alters mouse behavior
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Richard, S; Aguilera, N; Thevenet, M; Dkhissi-Benyahya, O; Flamant, F
- BEHAVIOURAL BRAIN RESEARCH
- Collaborative platform: PBES; Acknowledged platforms: PBES, PLATIM
Abstract :
In humans, alterations in thyroid hormone signalling are associated with mood and anxiety disorders, but the neural mechanisms underlying such association are poorly understood. The present study investigates the involvement of neuronal thyroid hormone receptor a (TR alpha) in anxiety, using mouse genetics and Cre/loxP technology to specifically alter TRa signalling in neurons. We evaluated the behaviour of mice expressing a dominant negative, neuron-specific mutation of TRa (TR alpha(AMI)/Cre3 mice), using the elevated plus maze, light-dark box and open-field tests. In a first experiment, mice were housed individually, and the behaviour of TR alpha(AmI)/Cre3 mice differed significantly from that of control littermates in these 3 tests, suggesting heightened anxiety. In a second experiment, designed to evaluate the robustness of the results with the same 3 tests, mice were housed in groups. In these conditions, the behaviour of TR alpha(AmI)/Cre3 mice differed from that of control littermates only in the light-dark box. Thus, TR alpha(AMI)/Cre3 mice appear to be more likely to develop anxiety under stressful housing conditions than control mice. These results suggest that in adult mice, thyroid hormone signalling in neurons, via TRa, is involved in the control of anxiety behaviour.
MAR 2017
DOI : 10.1016/j.bbr.2016.12.025
Pubmed : 28011173
- Structural basis of homo- and heterotrimerization of collagen I
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Sharma, U; Carrique, L; Vadon-Le Goff, S; Mariano, N; Georges, RN; Delolme, F; Koivunen, P; Myllyharju, J; Moali, C; Aghajari, N; Hulmes, DJS
- NATURE COMMUNICATIONS
- Collaborative platform: PSF; Acknowledged platform: PSF
Abstract :
Fibrillar collagen molecules are synthesized as precursors, procollagens, with large propeptide extensions. While a homotrimeric form (three alpha 1 chains) has been reported in embryonic tissues as well as in diseases (cancer, fibrosis, genetic disorders), collagen type I usually occurs as a heterotrimer (two alpha 1 chains and one alpha 2 chain). Inside the cell, the role of the C-terminal propeptides is to gather together the correct combination of three a chains during molecular assembly, but how this occurs for different forms of the same collagen type is so far unknown. Here, by structural and mutagenic analysis, we identify key amino acid residues in the alpha 1 and alpha 2 C-propeptides that determine homo-and heterotrimerization. A naturally occurring mutation in one of these alters the homo/heterotrimer balance. These results show how the C-propeptide of the alpha 2 chain has specifically evolved to permit the appearance of heterotrimeric collagen I, the major extracellular building block among the metazoa.
MAR 2017
DOI : 10.1038/ncomms14671
Pubmed : 28281531
- A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization
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Lynch, B; Bancelin, S; Bonod-Bidaud, C; Gueusquin, JB; Ruggiero, F; Schanne-Klein, MC; Allain, JM
- ACTA BIOMATERIALIA
- Acknowledged platform: PBES
Abstract :
Skin is a complex, multi-layered organ, with important functions in the protection of the body. The dermis provides structural support to the epidermal barrier, and thus has attracted a large number of mechanical studies. As the dermis is made of a mixture of stiff fibres embedded in a soft non-fibrillar matrix, it is classically considered that its mechanical response is based on an initial alignment of the fibres, followed by the stretching of the aligned fibres. Using a recently developed set-up combining multiphoton microscopy with mechanical assay, we imaged the fibres network evolution during dermis stretching. These observations, combined with a wide set of mechanical tests, allowed us to challenge the classical microstructural interpretation of the mechanical properties of the dermis: we observed a continuous alignment of the collagen fibres along the stretching. All our results can be explained if each fibre contributes by a given stress to the global response. This plastic response is likely due to inner sliding inside each fibre. The non-linear mechanical response is due to structural effects of the fibres network in interaction with the surrounding non-linear matrix. This multiscale interpretation explains our results on genetically-modified mice with a simple alteration of the dermis microstructure. Statement of Significance Soft tissues, as skin, tendon or aorta, are made of extra-cellular matrix, with very few cells embedded inside. The matrix is a mixture of water and biomolecules, which include the collagen fibre network. The role of the collagen is fundamental since the network is supposed to control the tissue mechanical properties and remodeling: the cells attach to the collagen fibres and feel the network deformations. This paper challenges the classical link between fibres organization and mechanical properties. To do so, it uses multiscale observations combined to a large set of mechanical loading. It thus appears that the behaviour at low stretches is mostly controlled by the network structural response, while, at large stretches, the fibre inner-sliding dominate.
MAR 2017
DOI : 10.1016/j.actbio.2016.12.051
Pubmed : 28043893
- Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites
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Boson, B; Denolly, S; Turlure, F; Chamot, C; Dreux, M; Cosset, FL
- GASTROENTEROLOGY BACKGROUND & AIMS
- Collaborative platform: PLATIM; Acknowledged platform: PLATIM
Abstract :
Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. METHODS: Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. RESULTS: Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. CONCLUSIONS: In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.
MAR 2017
DOI : 10.1053/j.gastro.2016.11.047
Pubmed : 27932311
- miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling
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El Helou, R; Pinna, G; Cabaud, O; Wicinski, J; Bhajun, R; Guyon, L; Rioualen, C; Finetti, P; Gros, A; Mari, B; Barbry, P; Bertucci, F; Bidaut, G; Harel-Bellan, A; Birnbaum, D; Charafe-Jauffret, E; Ginestier, C
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CELL REPORTS
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Acknowledged platform: Vectorology
Abstract :
Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define this state are unclear. We have performed two microRNA (miRNA) gain-and loss-of-function screens to identify miRNAs that regulate the choice between bCSC self-renewal and differentiation. We find that micro-RNA (miR)-600 silencing results in bCSC expansion, while its overexpression reduces bCSC self-renewal, leading to decreased in vivo tumorigenicity. miR-600 targets stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. In the absence of miR-600, WNT signaling is active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT and promotes bCSC differentiation. In a series of 120 breast tumors, we found that a low level of miR-600 is correlated with active WNT signaling and a poor prognosis. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decisions and influences tumor progression.
FEB 2017
DOI : 10.1016/j.celrep.2017.02.016
Pubmed : 28249169
- Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes
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Jean, A; Tardy, F; Allatif, O; Grosjean, I; Blanquier, B; Gerlier, D
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PLOS ONE
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Collaborative platform: AGC; Acknowledged platform: BSL3
Abstract :
Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination.
FEB 2017
DOI : 10.1371/journal.pone.0172358
Pubmed : 28225826
- LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription
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Scionti, I; Hayashi, S; Mouradian, S; Girard, E; de Lima, JE; Morel, V; Simonet, T; Wurmser, M; Maire, P; Ancelin, K; Metzger, E; Schule, R; Goillot, E; Relaix, F; Schaeffer, L
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CELL REPORTS
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Acknowledged platform: PBES
Abstract :
MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.
FEB 2017
DOI : 10.1016/j.celrep.2017.01.078
Pubmed : 28228264
- Quantification of Detergents Complexed with Membrane Proteins
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Chaptal, V; Delolme, F; Kilburg, A; Magnard, S; Montigny, C; Picard, M; Prier, C; Monticelli, L; Bornert, O; Agez, M; Ravaud, S; Orelle, C; Wagner, R; Jawhari, A; Broutin, I; Pebay-Peyroula, E; Jault, JM; Kaback, HR; le Maire, M; Falson, P
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SCI REP
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Collaborative platform: PSF
Abstract :
Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.
FEB 2017
DOI : 10.1038/srep41751
Pubmed : 28176812
- Evaluation of Three Devices for the Isolation of the Stromal Vascular Fraction from Adipose Tissue and for ASC Culture: A Comparative Study
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Rodriguez, J; Pratta, AS; Abbassi, N; Fabre, H; Rodriguez, F; Debard, C; Adobati, J; Boucher, F; Mallein-Gerin, F; Auxenfans, C; Damour, O; Mojallal, A
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STEM CELLS INTERNATIONAL
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Acknowledged platform: AGC
Abstract :
Adipose-derived stem/stromal cells (ASCs) reside in the stromal vascular fraction (SVF) of adipose tissue (AT) and can be easily isolated. However, extraction of the SVF from lipoaspirate is a critical step in generating ASC, and semiautomated devices have been developed to enhance the efficacy and reproducibility of the outcomes and to decrease manipulation and contamination. In this study, we compared the reference method used in our lab for SVF isolation from lipoaspirate, with three medical devices: GID SVF-1 (TM), Puregraft (TM), and Stem. pras (R). Cell yield and their viability were evaluated as well as their phenotype with flow cytometry. Further on, we determined their proliferative potential using population doublings (PD), PD time (PDT), and clonogenicity assay (CFUF). Finally, we checked their genetic stability using RT-qPCR for TERT mRNA assay and karyotyping as well as their multilineage potential including adipogenic, chondrogenic, and osteogenic differentiation. Our results demonstrate that all the devices allow the production of SVF cells with consistent yield and viability, in less time than the reference method. Expanded cells from the four methods showed no significant differences in terms of phenotype, proliferation capabilities, differentiation abilities, and genetic stability.
JAN 2017
DOI : 10.1155/2017/9289213
Pubmed : 28321259