Les livres français pour la jeunesse traduits à l'étranger : esquisses d'une enquête

International audienceEnquête menée avec Nathalie Beau (La Joie par les Livres - IBBY France) sur les livres pour la jeunesse français contemporains traduits à l'étranger

Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

International audienceIn Brief 20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled pro-teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFN␥ or amplified in a 20% O 2 environment. Graphical Abstract Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O 2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity

Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during <i>Drosophila</i> hematopoiesis

<div><p>A tight regulation of transcription factor activity is critical for proper development. For instance, modifications of RUNX transcription factors dosage are associated with several diseases, including hematopoietic malignancies. In <i>Drosophila</i>, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). However, the mechanism of action of this conserved family of proteins involved in leukemia remains largely unknown. Here we further characterized MLF’s mode of action in <i>Drosophila</i> blood cells using proteomic, transcriptomic and genetic approaches. Our results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and we demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, <i>dnaj-1</i> loss causes an increase in Lz⁺ blood cell number and size similarly as in <i>mlf</i> mutant larvae. Moreover we find that <i>dnaj-1</i> genetically interacts with <i>mlf</i> to control Lz level and Lz⁺ blood cell development <i>in vivo</i>. In addition, we show that <i>mlf</i> and <i>dnaj-1</i> loss alters Lz⁺ cell differentiation and that the increase in Lz⁺ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, using different conditions to manipulate Lz activity, we show that high levels of Lz are required to repress <i>Notch</i> transcription and signaling. All together, our data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of <i>Notch</i> expression and signaling to prevent aberrant blood cell development. Thus our findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development <i>in vivo</i>.</p></div

MLF and DnaJ-1 bind Lz and control its stability and activity.

<p>(A) Luciferase assays in Kc167 cells treated with the indicated dsRNA and transfected with 4xPPO2-Fluc reporter plasmid in the presence or not (ctr) of the pAc-Lz-V5 expression plasmid. pAc-Rluc was used as an internal normalization control. (B) Western blots showing Lz-V5, MLF, <i>Renilla</i> luciferase (R luc) and Tubulin (Tub) expression in Kc167 cells treated with the indicated dsRNA and cotransfected with pAc-Lz-V5 and pAc-Rluc expression vectors. (A, B) dsDnaJ-1 (a) and (b) correspond to two distinct dsRNAs targeting <i>dnaj-1</i>. Of note, the multiple bands for Lz are only observed using C terminally (V5) tagged versions of Lz and not with N terminally (GFP) tagged Lz; they likely represent internal translation initiation events. The multiple bands observed using a MLF antibody could represent different MLF protein isoforms as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006932#pgen.1006932.ref017" target="_blank">17</a>]. (C, D) Luciferase assays (C) and Western blots (D) performed on Kc167 cells transfected with the 4xPPO2-Fluc reporter plasmid and pAc-based expression plasmids for Lz and for different DnaJ-1 variants as indicated. Rluc and Tubulin were used as internal controls. (E, F) Western blots showing the results of immunoprecipitation experiments against GFP performed in Kc167 cells transfected with expression vectors for HA-MLF (E) or HA-DnaJ-1 (F) and GFP or GFP-Lz as indicated in the upper part of the panels. (G, H) Western blots showing the results of immunoprecipitation experiments against GFP performed in Kc167 cells transfected with expression vectors for GFP-Lz and various HA-MLF (G) or HA-DnaJ-1 (H) mutants. (A, C) For luciferase assays means and standard deviations of results from biological triplicates are shown. ***: p-value<0.001, **: p-value<0.01 (Students t-tests) as compared to Lz with dsGFP condition.</p

High levels of MLF prevent Lz degradation in the absence of DnaJ-1.

<p>(A-C) Fluorescent immunostainings of Lz in circulating blood cells from <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> control (A), <i>dnaj1</i>^<i>-/-</i> (B) and <i>mlf</i>^<i>-/-</i> (C) third instar larvae. (D) Corresponding quantifications of Lz protein level. (E-H) Immunostainings against Lz (red) and HA-MLF (green) in Kc167 cells treated with the indicated dsRNA and transfected with pAc-Lz-V5 alone (E, G) or in combination with pAc-3HA-MLF (F, H). (I) Corresponding quantification of Lz levels in Kc167 cells. (J-M) Immunostainings against Lz in circulating blood cells from <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> control (J), <i>UAS-dsMLF</i> (K), <i>dnaj1</i>^<i>-/-</i> (L) and <i>UAS-dsMLF</i>; <i>dnaj1</i>^<i>-/-</i> (M) third instar larvae. (N) Corresponding quantification of Lz protein levels in lz>GFP⁺ larval blood cells. (A-C, E-H, J-M) Nuclei were stained with Topro3. Lz staining only is shown in the lower panels. Scale bar: 10 μm. (O, P) Relative lz>GFP⁺ blood cell number (O) and size (P) in <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> third instar larvae of the indicated genotypes. (D, I, N-P) *: p-value<0.05, **: p-value<0.01, ***: p-value<0.001.</p

The increase in lz>GFP⁺ cell number and size in <i>mlf</i> and <i>dnaj-1</i> mutant larvae is caused by overactivation of the Notch signaling pathway.

<p>(A-C) Immunostainings against Notch (NECD: Notch extracellular domain) in blood cells from <i>lz-GAL4</i>,<i>UAS-mCD8-GFP/+</i> control (A), <i>mlf</i>^<i>-/-</i> (B) and <i>dnaj-1</i>^<i>-/-</i> (C) larvae. The immunostaining against Notch protein only is shown in the lower panels. Nuclei were stained with Topro3. (D) Quantification of NECD immunostainings in lz>GFP⁺ and lz>GFP^- blood cells from control, <i>mlf</i>^<i>-/-</i> and <i>dnaj-1</i>^<i>-/-</i> larvae. (E) Quantification of NICD (Notch intracellular domain) immunostainings in lz>GFP⁺ blood cells from control, <i>mlf</i>^<i>-/-</i> and <i>dnaj-1</i>^<i>-/-</i> larvae. (F-H) Expression of the Notch pathway reporter Klu-Cherry in lz>GFP⁺ blood cells from control, <i>mlf</i>^<i>-/-</i> or <i>dnaj-1</i>^<i>-/-</i> larvae. Klu-Cherry expression only is shown in the lower panels. (I) Corresponding quantification of Klu-Cherry level. (J) Quantification of the expression level of the Notch pathway reporter NRE-GFP in PPO1-expressing cells from control, <i>mlf</i>^<i>-/-</i> or <i>dnaj-1</i>^<i>-/-</i> larvae. (K, L) Relative lz>GFP⁺ blood cell number (K) and size (L) in third instar larvae of the indicated genotypes.</p

<i>dnaj-1</i> controls crystal cell development.

<p>(A, B) Quantification of circulating lz>GFP⁺ cell number (A) and lz>GFP⁺ or lz>GFP^- cell size (B) in <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> third instar larvae of the indicated genotypes. (C-E) Fluorescent immunostainings of the crystal cell differentiation marker PPO1 in third instar lz>GFP⁺ hemocytes. The right panels show PPO1 immunostaining only. Nuclei were stained with Topro3. Scale bar: 10 μm. (F-H) Bright field images of the posterior segments of third instar larvae heat-treated at 65°C for 10 min to induce crystal cell melanization. (I, J) Relative lz>GFP⁺ blood cell number (I) and size (C) in <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> third instar larvae of the indicated genotypes. (A, B, I, J) *:p-value<0.05, **: p-value<0.01 and ***: p-value<0.001 compared to control.</p

First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier

International audienceMassive proliferation of some toxic marine dinoflagellates is responsible for the occurrence of harmful algal blooms and the contamination of fish and shellfish worldwide. Pinnatoxins (PnTx) (A-H) comprise an emerging phycotoxin family belonging to the cyclic imine toxin group. Interest has been focused on these lipophilic, fast-acting and highly potent toxins because they are widely found in contaminated shellfish, and can represent a risk for seafood consumers. PnTx display a potent antagonist effect on nicotinic acetylcholine receptors (nAChR), and in this study we assessed in vivo the ability of PnTx-G to cross physiological barriers to reach its molecular target. Radiolabeled [3H]-PnTx-G synthesized with good radiochemical purity and yield retained the high affinity of the natural toxin. Oral gavage or intravenous administration to adult rats and digital autoradiographic analyses revealed the biodistribution and toxicokinetics of [3H]-PnTx-G, which is rapidly cleared from blood, and accumulates in the liver and small intestine. The labeling of peripheral and brain adult/embryo rat tissues highlights its ability to cross the intestinal, blood-brain and placental barriers. High-resolution 3D-imaging and in vitro competition studies on rat embryo sections revealed the specificity of [3H]-PnTx-G binding and its selectivity for muscle and neuronal nAChR subtypes (such as α7 subtype). The use of a human perfused cotyledon model and mass spectrometry analyses disclosed that PnTx-G crosses the human placental barrier. The increasing worldwide occurrence of both the dinoflagellate Vulcanodinium rugosum and PnTx-contaminated shellfish, due to climate warming, raises concerns about the potential adverse impact that exposure to pinnatoxins may have for human health

Lz represses <i>Notch</i> expression.

<p>(A-D) Immunostainings against NECD (Notch extracellular domain) in blood cells from <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+</i> (A), <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/Y</i> (B), <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/Y; UAS-lz</i> (C) and <i>lz-GAL4</i>, <i>UAS-mCD8-GFP/+; UAS-lz</i> (D) third instar larvae. NECD immunostaining only is shown in the lower panels. Nuclei were stained with Topro3. (E) Corresponding quantifications of NECD in lz>GFP⁺ blood cells. (F-F”‘) Immunostaining against Lz in circulating blood cells from <i>Notch</i>^{<i>GMR30A01</i>}<i>-GAL4</i>, <i>UAS-GFPnls</i> third instar larvae. Nuclei were stained with Topro3. (F’-F”‘): single channel images. (G-J) <i>Notch</i>^{<i>GMR30A01</i>}<i>-GAL4</i>-driven expression of GFP in circulating blood cells from larvae of the indicated genotypes. (K) Corresponding quantifications of the level of GFP. (A-D, F-J) Scale bar: 10μm. (E, K) *: p-value<0.05, *** p-value<0.001.</p