Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems

Involvement of Nuclear Factor of Activated T-Cells (NFAT) transcription factors during human primary myoblast differentiation

Calcium signaling plays key roles during human myoblast differentiation. The calcium-dependent phosphatase Calcineurin is essential for myoblast differentiation and muscle regeneration. NFAT transcription factors (Nuclear Factor of Activated T-cell) are the major Calcineurin targets. By investigating their expression and their role, I found that three NFAT are present in human primary myoblasts, NFATc1, NFATc3 and NFATc4. Surprisingly, while their expression increased during differentiation, NFATc1 is more expressed in myotubes and NFATc4 in the reserve cells. Although NFATc3 is expressed in both cell types but does not seem to have a particular role during myoblast differentiation. When NFATc1 or NFATc4 expression is impaired by siRNAs, differentiation is affected. The expression of late differentiation markers, Myosin HC and STIM1L, is decreased and myotube formation is impaired, but differently. Indeed, NFATc1 knockdown strongly reduced the number and the surface of myotubes formed, NFATc4 knockdown increased myotube surface and reduced the reserve cells pool

Distinct roles of NFATc1 and NFATc4 in human primary myoblast differentiation and in the maintenance of reserve cells

Ca2+ signaling plays a key role during human myoblast differentiation. Among Ca2+-sensitive pathways, calcineurin is essential for myoblast differentiation and muscle regeneration. Nuclear factor of activated T-cell (NFAT) transcription factors are the major calcineurin targets. We investigated the expression and the role of each NFAT gene during human primary myoblast differentiation. We found that three NFAT isoforms are present, NFATc1, NFATc3 and NFATc4. Importantly, while their mRNA expression increases during differentiation, NFATc1 is more highly expressed in myotubes, whilst NFATc4 is specifically maintained in reserve cells. NFATc3 is present in both cell types, although no specific role during myoblast differentiation was observed. Knockdown of either NFATc1 or NFATc4 affects the differentiation process similarly, by decreasing the expression of late differentiation markers, but impairs myotube formation differently. Whereas NFATc1 knockdown strongly reduced the number and the surface area of myotubes, NFATc4 knockdown increased the surface area of myotubes and reduced the pool of reserve cells. We conclude that NFAT genes have specific roles in myotube formation and in the maintenance of the reserve cell pool during human postnatal myogenesis

Epidermal growth factor receptor down-regulation triggers human myoblast differentiation

Initiation of human myoblast differentiation requires a negative shift (hyperpolarization) of the resting potential of myoblasts that depends on the activation of Kir2.1 potassium channels. These channels are regulated by a tyrosine phosphorylation. Using human primary myoblast culture, we investigated a possible role of various receptor tyrosine kinases in the induction of the differentiation process. We found that Epidermal Growth Factor Receptor (EGFR) is a key regulator of myoblast differentiation. EGFR activity is down-regulated during early human myoblast differentiation, and this event is required for normal differentiation to take place. Furthermore, EGFR silencing in proliferation conditions was able to trigger the differentiation program. This occurs through an increase of Kir2.1 channel activity that, via a rise of store-operated Ca(2+) entry, leads to the expression of myogenic transcription factors and muscle specific proteins (Myogenin, Myocyte Enhancer Factor 2 (MEF2), Myosin Heavy Chain (MyHC)). Finally, blocking myoblast cell cycle in proliferation conditions using a cdk4 inhibitor greatly decreased myoblast proliferation but was not able, on its own, to promote myoblast differentiation. Taken together, these results show that EGFR down-regulation is an early event that is required for the induction of myoblast differentiation

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors

International audienc

Kir2.1 is activated by EGFR knockdown in myoblasts.

<p>Myoblasts were transfected with either control siRNA or siEGFR. <b>A</b>. Percentages of transfected myoblasts with functional Kir2.1 channels, 48 hours post-transfection. <b>B.</b> Current densities of the total population of myoblasts (including myoblasts with no current, i.e. <5 pA). <b>C.</b> Current-voltage relationships of a myoblast transfected with siEGFR. Voltage-steps were to −40, −60, −80, −100, −120 and −140 mV from a holding potential at −60 mV. The inset shows a control myoblast with no Kir2.1 current, a typical Kir2.1 current recorded from a myoblast, 48 h after transfection with siEGFR. Addition of 500 µM Ba²⁺ inhibited this current. <b>D.</b> Myoblasts were first transfected with control siRNA or siEGFR, and 24 h later with a plasmid coding for GFP-Kir2.1. One day after, immunoprecipitation of GFP was performed. Immunoblots reveal Kir2.1 and phospho-tyrosine (P-Tyr). <b>E</b>. Cytoplasmic Ca²⁺ was assessed with Fura-2-AM on proliferating myoblasts, 2 days after siRNA transfection. Intracellular Ca²⁺ stores were depleted with 10 µM thapsigargin (Tg) in a medium containing 250 nM Ca²⁺. Then 1.8 mM Ca²⁺ was subsequently added to reveal SOCE. The first part of the experiment was performed with a medium containing 30 mM KCl in order to clamp cells at around −40 mV. The second part was performed with a medium containing 5 mM KCl allowing cells to hyperpolarize. <b>F</b>. Quantification of peak SOCE (n = 6; * p<0.05).</p

Modulation of EGFR activity during the first hours of myoblast differentiation.

<p>All results were normalized to the result obtained for GM conditions. <b>A</b>. Myoblasts were lysed in proliferation (GM) and at different times in differentiation conditions (DM). Western blot analysis of EGFR on whole cells extracts. EGFR expression decreases by 64% after 24 h in DM (n = 5). <b>B</b>. Myoblasts were fixed in proliferation (GM) and at different times in differentiation conditions (DM). Flow cytometry was performed on non-permeabilized myoblasts. For the control condition myoblasts were only incubated with the secondary antibody. A significant decrease of EGFR at the plasma membrane is observed after 9 h in DM (n = 5). To control the specificity of the antibody against EGFR, we verified that the antibody did not bind any more to myoblasts transfected with a siEGFR. <b>C-D-E</b>. Total EGFR, phospho-p42/p44 MAPK and Myogenin expressions were assessed by Western blot. α-Tubulin expression was used as a loading control. <b>C.</b> Efficiency of EGFR inhibitors. Myoblasts were cultured in GM for 1 h with AG1478 at 10 µM or PD153035 at 3 µM. Western blot analysis shows a significant decrease of phospho-p42/p44 MAPK expression but no difference of EGFR expression (all n = 3). <b>D</b>. Myogenin expression after 24 h treatment with EGFR inhibitors in proliferating conditions (n = 4). <b>E</b>. Phospho-p42/p44 MAPK and EGFR expression during the first hour of differentiation (n = 5).</p

Vitamin K3 prevents differentiation without inducing proliferation.

<p>A. Myoblasts were cultured either in GM or in DM ±10 µM vitamin K3. Differentiation was assessed by immunostaining (MEF2 in red; MyHC in green; nuclei in blue with DAPI). B. Quantification of the immuno-fluorescence shown in A (n = 4). C. Vitamin K3 in differentiation conditions for 24 h does not increase cell proliferation as assessed by the average of the number of nuclei (DAPI) per field (from 3 independent experiments; p = 0.4).</p

Myoblast differentiation is induced by EGFR knockdown.

<p>Proliferating myoblasts were transfected either with a control siRNA or with a siEGFR, and then maintained in proliferation condition for 5 days. <b>A.</b> Total cell lysates were analyzed by Western Blot. Myogenin and MEF2 expressions were used as early markers of differentiation, Myosin heavy chain (MyHC) as a late marker of differentiation, and α-Tubulin as a loading control. <b>B</b>. Differentiation of myoblasts was observed by the staining of MEF2 (red) and MyHC (green), and nuclei by DAPI (blue). Cesium (Cs 10 mM) was added to the medium to block the hyperpolarization. Myotubes resulting from myoblast fusion were observed (white arrows). <b>C.</b> Quantification of the immuno-fluorescence shown in B (n>3).</p

EGFR expression is down-regulated during myoblast differentiation.

<p>Cells were cultured in either growth medium (GM) or differentiation medium (DM) for 24 h. <b>A</b>. Phospho-RTK arrays were hybridized with 500 µg proteins of whole cell lysates and detected with an anti-phosphotyrosine antibody. Quantification of each spot was performed and expressed in arbitrary units (AU). The results for the ErbB receptors are shown (n = 2). <b>B</b>. EGFR and CD56 expression using FACS technique. Myoblasts were fixed and non-permeabilized (n = 5). <b>C.</b> EGFR expression was confirmed by Western Blot (whole cell lysates). Myogenin and MEF2 expressions were used as differentiation markers, and α-Tubulin as loading control. <b>D</b>. Efficiency of the siRNA against EGFR. Myoblasts were transfected either with a control siRNA or with a siEGFR, and then maintained in proliferation condition for 24 h. EGFR expression was determined by Western Blot (a representative result is shown, n>5).</p