10 research outputs found
L'ossigenoterapia: Indicazioni, vie di somministrazione e parametri da valutare
The main issues concerning oxygen treatment in children are discussed in this paper ranging from physiopathological premises to practical daily issues. Indications to oxygen treatment, monitoring issues, clinical tips, advantages and disadvantages of different ways of administering oxygen are focused, mainly with a practice based approach. Signs and symptoms of hypoxemia, hypercapnia and increased breathing work are highlighted. The two different mechanisms leading to respiratory failure (type I and type II) are discussed along with the proper indications for treatment
miR-204 Targeting of <i>Ankrd13A</i> Controls Both Mesenchymal Neural Crest and Lens Cell Migration
<div><p>Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of <i>in vivo</i> and <i>in vitro</i> approaches, we demonstrate that these actions are mediated by the direct targeting of the <i>Ankrd13A</i> gene, which in turn controls focal cell adhesion formation and distribution. Significantly, <i>in vivo</i> restoration of abnormally elevated levels of <i>Ankrd13A</i> resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time <i>in vivo,</i> the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a “master regulator” of the molecular networks that regulate lens morphogenesis in vertebrates.</p></div
miR-204 directly targets <i>Ankrd13A</i>.
<p>(A–D) Frontal sections of St24 and St28 WT medaka embryos hybridized in single whole-mount RNA ISH with probes against miR-204 (A, C) and <i>olAnkrd13A</i> (B, D). (E) The predicted target site of miR-204 within the 3′-UTR of the <i>Ankrd13A</i> gene in different species: conserved nucleotides are marked in red and non-conserved nucleotides are marked in black. (F) Histograms showing the fold change variations (expressed as 2<sup>−ΔΔCt</sup> values) in the mRNA levels of <i>Ankrd13A</i> quantified by qRT-PCR in medaka embryos injected with Mo-miR-204 or miR-204 with respect to controls. The <i>Hprt</i> and <i>Gapdh</i> genes were used to normalize the results. (G) Relative Luc activities in H36CE cells as fold differences in the Luc/Renilla ratios normalized to the value of Luc reporter constructs. miR-204 addition significantly decreases Luc activity of the construct containing the 3′-UTR of <i>ANKRD13A</i> when compared to controls. ***P<0.001 (t tests). Three point mutations in the predicted miR-204 target site in <i>ANKRD13A</i> inhibit this effect (no significant variation when compared with the thymidine kinase (TK)- Luc control). (H) Immunofluorescence with a monoclonal antibody against the FLAG tag (red) after short transfection times of FLAG-tagged full-length <i>ANKRD13A</i> to minimize overexpression. Boxed area is magnified in I–K. (I–K) Actin was visualized by staining with phalloidin (Red). ANKRD13A (Green) colocalizes with actin filaments (arrows). Notably, the ANKRD13A OE determined the depolymerization of actin filaments.</p
miR-204 controls mesenchymal NCC migration <i>in vivo</i>.
<p>(A–F) Expression of the <i>olTrpm1</i>:GFP transgene in migrating NCC of control, miR-204 MO, and miR-204 OE medaka embryos. Representative images from time-lapsed movie of <i>olTrpm1</i>:GFP expression in the trunk from st24 (A, B, C) to St25 (D, E, F; 8 hrs later), dorsal view (see also Movies in the supplementary material). (G–I) Time-lapse analysis showing cellular protrusion of migrating NCCs in control (G), miR-204 MO (H), and miR-204 OE (I) medaka embryos. Notably, Mo-miR-204 induces a shorter cellular protrusion (H), whereas miR-204 OE NCCs show longer protrusions (I) when compared to control embryos (G). (J) Tracking the position of the migrating NCCs revealed that the velocity of NCC migration was decreased in MO and increased in miR-204 OE embryos. (K) Analysis of maximum extension of the cellular protrusion of migrating NCCs revealed that the projection cell lengths were decreased in MO and increased in miR-204 OE embryos. Bars in J and K are means ± SEM of values; ***P<0.0001 (t tests). Images are representative of at least 5 time-lapse movies from 10 independent experiments.</p
Ankrd13A depletion specifically rescues the miR-204 morphant lens phenotype.
<p>Frontal sections of St24 control (A), MO-miR-204 (B), and MO-miR-204/Mo-Ankrd13A (C)-injected medaka embryos processed for whole-mount RNA ISH with an <i>olPax6</i> probe (A–C). Notably, interference with <i>Ankrd13A</i> expression fully recovers primary fiber lens cell mislocalization, but not lens epithelial cell localization that are dorsally located. Dashed lines mark the boundaries between the lens epithelial monolayer and the primary fiber cells. Bright-field stereomicroscopy images of St40 control (D,G), Mo-miR-204 (E,H), Mo204/Mo-Ankrd13A (F,I) injected medaka embryos as dorsal (D, E, F), and lateral (G, H, I) views. In Mo-miR-204/Mo-Ankrd13A-injected embryos, the lens phenotype is rescued. Notably, the lens does not protrude out of the optic cup (F, I, yellow arrows) when compared to miR-204 morphant embryos (E, H, red arrows).</p
ANKRD13A controls cytoskeleton and FA organization.
<p>(A–B) Immunostaining of FAK (green) and nuclei (DAPI-blue) in ANKRD13A-3×Flag (Red) OE H36CE lens cells. (C–C′) Immunostaining of β-tubulin (green) and nuclei (DAPI-Blu) in ANKRD13A-3×Flag (Red) OE H36CE lens cells. Immunostaining of β-tubulin (green), nuclei (DAPI-blu) and actin filaments with phalloidin (Red) in control (D–F), and ANKRD13A KD (G–I) H36CE lens cells. (J) Relative percentage of FA per area in H36CE cells. ANKRD13A OE displays a significant increase in the number of FA per area with respect to control. A marked decrease of both percentage of FA per area and FA polarized distribution was observed in ANKRD13A KD H36CE lens cells. ***P<0.0001 (t tests). (K) Extent of cell adhesion was plotted as a percentage. The number of cell adhesions is significantly increased by ANKRD13A OE and significantly decreased by ANKRD13A KD in H36CE lens cells. ***P<0.0001 (t tests).</p
Schematic model of the proposed function of miR-204 in lens development.
<p>MiR-204 acts in a context-dependent manner in proliferating epithelial, migrating, and differentiating lens cells. In epithelial cells, miR-204 controls the expression of the <i>Runx2</i> gene, contributing to the regulation of the proliferative pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061099#pone.0061099-Hoffmann2" target="_blank">[50]</a>. In migrating cells, miR-204 activity regulates fiber lens cell migration by targeting <i>Ankrd13A</i> gene and modulating FA and cytoskeleton organization (this paper). In differentiating lens cells, miR-204 modulates the <i>Meis2/Pax6</i> gene pathways and the related cell differentiation molecular networks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061099#pone.0061099-Conte1" target="_blank">[13]</a>.</p
miR-204 affects cytoskeleton and FA organization.
<p>Immunostaining of actin filaments (Phalloidin, red), FA (Fak, green) and nuclei (DAPI-blue) in control (A, D), miR-204 KD (B, E), and miR-204 OE (C, F) H36CE lens cells. Immunostaining of β-tubulin (green) and nuclei (Propidium Iodide (PI), red) in control (G), miR-204 KD (H), and miR-204 OE (I) H36CE lens cells. (J) miR-204 depletion significantly increases the number of FA (indicated as percentage/mm<sup>2</sup>) in H36CE cells with respect to control. A marked decrease of both percentage of FA per area and FA polarized distribution was observed in miR-204 OE H36CE lens cells. ***P<0.0001 (t tests). K Extent of cell adhesion was plotted as a percentage. miR-204 OE significantly decrease the number of cell adhesion. A marked increase of this percentage was observed in miR-204 KD H36CE lens cells. ***P<0.0001 (t tests).</p