De novo mutations in SMCHD1 cause Bosma arhinia microphthalmia syndrome and abrogate nasal development

Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD

Effets de lactobacillus casei sur le système immunitaire muqueux digestif de l'enfant

L'objectif de ce travail est de caractériser l'influence d'une alimentation contenant une souche probiotique sur la réponse immunitaire muqueuse digestive constituée par les sIgA. Ce marqueur est du plus grand intérêt pour toutes les études sur la vaccination orale, la tolérance orale, la prévention des intoxications alimentaires, ou l'allergie alimentaire. Au niveau de la muqueuse intestinale, le rôle de défense de l'organisme est principalement joué par la flore microbienne et par le système immunitaire muqueux, via les IgA sécrétoires totales qui représentent environ 20 mg pour 100 g de selles chez de jeunes enfants sains. Les bactéries lactiques probiotiques, telles que L casei tendent à faire diminuer les taux de sIgA fécales, ainsi que ceux de TGF-b. Cette cytokine, sécrétée par les cellules de type Th2 et Th3, est un facteur de commutation de classe et permet la synthèse d'IgA par les cellules B au niveau de la muqueuse intestinale. D'autre part, une supplémentation en bactéries lactiques (L bulgaricus et S thermophilus et/ou L casei) augmente les taux d'IgA spécifiques des allergènes alimentaires (gliadine, arachide, caséine, alpha-lactalbumine et b-lactoglobuline) et d'IL-10 dans les selles d'enfants sains. Ce phénomène ne se poursuit après la supplémentation que dans le cas d'une consommation de L casei. Cette interleukine joue un rôle dans le phénomène de tolérance orale qui consiste en une inhibition des réponses immunitaires systémiques (notamment à médiation cellulaire: type Th1), tout en développant des réponses immunitaires humorales muqueuses, ce qui explique l'augmentation des taux d'IgA spécifiques des allergènes alimentaires étudiés. Le phénomène de tolérance orale s'applique aux bactéries de la flore intestinale ainsi qu'aux probiotiques. La présence de L casei dans l'alimentation provoque une augmentation des taux d'IgA spécifiques de certains épitopes de L casei. L'identification de ces derniers montre que L casei possède cinq épitopes immunogènes majeurs de 31, 35, 39, 51 et 72 kDa et deux molécules de 14 et 55 kDa ne présentant pas cette propriété immunogène.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Signaling <i>via</i> Class IA Phosphoinositide 3-Kinases (PI3K) in Human, Breast-Derived Cell Lines

<div><p>We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P₃-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN^−/− cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN^−/− MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, <i>via</i> acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.</p></div

Relative expression of class I PI3Ks in breast-derived cell lines.

<p><b>Panel A</b>. MCF10a cells were cultured in full growth medium and then lysed and processed to perform mRNA-seq analysis. The corrected number of mapped sequence reads from p110α, p110β, p110δ and p110γ mRNAs are presented in a scatter plot based on 2 independent preparations of mRNA. <b>Panel B</b>. The indicated cells were grown in full growth medium and lysed and processed to quantify their relative expression of p110α, β and δ by immuno-blotting and use of fluorescent 2° antibodies and Li-Cor imaging. Actin was used as a loading control. The upper part of the figure shows a representative immuno-blot used to compile the data shown in the lower part of the figure. The data presented in the graph are means ± SE (n = 3 experiments).</p

Role of class IA PI3Ks in EGF-stimulated chemokinesis in MDA-MB 231 cells. Panel A.

<p>Individual MDA-MB 231 cells were tracked moving on Matrigel in stable gradients of EGF (the concentration of EGF in the reservoir was 0 ng/ml (a), 15 ng/ml (b), 30 ng/ml (c) and 60 ng/ml (d)) or expressing sh-CT (e), sh-p110 (f) or treated with LY294002 (g) in Dunn chambers. The data are presented centre-zeroed with the source of EGF at the top. Directionality was analysed using Mathmatica and significant directionality is denoted by a grey vector and arrow. The number of individual tracks analysed is shown (n) and were collected from at least 3 independent experiments. <b>Panel B.</b> The total accumulated distances moved by individual cells in the experiments shown in panel A are shown, the data presented are means. Parental MDA-MB 231 cells (some parental cells were pretreated with 10 µM LY294002) or derivatives expressing either control or p110α-directed shRNAi constructs (3 sh-CT (N2, N3 and N4, see Methods, separate cell lines expressing each construct were used and the data derived were pooled for presentation as they were indistinguishable) and 2 sh-p110α -expressing (A1 and A2, 2 cell lines expressing the individual constructs were used and the data from were pooled for presentation) independent, selected populations which were in the range 80–90% eGFP +ve) were exposed to EGF gradients (30 ng/ml EGF in the reservoir) in Dunn chambers. The cell tracks and their directionality are shown as in panel A. The number of cells tracked (n) is indicated and were collected from at least 4 independent experiments. The total accumulated distances moved by individual cells in the experiments shown in panel A are shown, the data presented are means. The data for all control shRNAi constructs and all p110α-directed constructs were pooled separately to enable an overall comparison of their effects. Statistical comparisons were conducted as in Fig. 4C.</p

PI3Kβ is the dominant class IA PI3K required for EGF-stimulated PKB phosphorylation in MDA-MB 468 cells. Panel A.

<p>MDA-MB 468 cells were serum-starved, treated with inhibitors (inhibitor concentrations, from the left, were; A66, 6 µM; A66, 2 µM, A66, 0.5 µM; TGX221, 40 nM; IC87114, 1 µM; “mix”, A66, 6 µM+TGX221, 40 nM+IC87114, 1 µM; “mix”, except with 2 µM A66; PI103, 1 µM) or vehicle for 20 mins and phosphorylation of PKB was quantified by immuno-blotting (S473 using fluorescent 2°-antibodies, T308 using HRP-linked 2°-antibodies). A representative set of blots, with β-COP as a loading control, are shown. <b>Panel B.</b> Data from experiments like those in panel A measuring phosphorylation of S473-PKB were quantified (only data from experiments with 6 µM A66 both alone and as a part of the “mix” are shown) and normalized to the vehicle treated samples (“starved”). Data presented are means ± SE (n = 3, experiments). Statistical comparisons were conducted as in Fig. 3. <b>Panel C.</b> Phosphorylation of T308 was quantified with the same blots as those shown in panel B. <b>Panel D.</b> Experiments like those shown above except some cells were stimulated with EGF (0.1 ng/ml) or EGF plus inhibitors. Phosphorylation of S473-PKB was quantified by immuno-blotting, normalized for loading using β-COP and corrected between experiments by expressing the data as a proportion of samples that were EGF-stimulated but not treated with inhibitors. Data are presented as means ± SE (n = 3, experiments). <b>Panel E.</b> Phosphorylation of T308-PKB was quantified with the same blots as those shown in panel D.</p