12 research outputs found
Normal ribosomal RNA processing in Cirhin-deficient larvae.
<p>(<b>A</b>) Northern blots demonstrating equivalent levels of pre-rRNA species in duplicate samples of mismatch MO-injected and <i>cirh1a</i> MO-injected embryos. Dashes and letters correspond to presumed pre-rRNA species identified by probes 5’ETS, ITS1, and ITS2, while asterisks indicate unknown pre-rRNA species (schematized in panel B). The far right panel shows the results of blotting with an 18S rRNA probe, demonstrating total RNA loading. Mean 45S/18S rRNA ratios were not statistically different between treatment groups (p>0.05). (<b>B</b>) Schematic depicting presumed pathway of zebrafish ribosomal RNA processing; individual RNA species not drawn to scale (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077670#B32" target="_blank">32</a>]).</p
Liver histology and ultrastructure in Cirhin-deficient larvae.
<p>(<b>A</b>-<b>F</b>) Liver histology of wild-type (A-C) and Cirhin-deficient (D-F) larvae is indistinguishable, although liver size and yolk consumption are decreased in Cirhin-deficient larvae as a result of mild developmental delay (D-E). Insets in B-C, E-F are high-magnification views of associated panel. (<b>G</b>-<b>L</b>) Transmission electron microscopy of wild-type (G-I) and Cirhin-deficient (J-L) larvae. Compared to wild-type, Cirhin-deficient hepatocytes have increased rough endoplasmic reticulum (J, red asterisk) and occasional cytoplasmic lamellations consistent with bile (K-L, black arrowheads). Biliary cells are outlined by red dashed lines and appear normal (H,K).</p
Zebrafish <i>cirh1a</i> is expressed in the developing liver and anterior gastrointestinal tract.
<p>(<b>A</b>-<b>C</b>) Whole-mount in situ hybridization for <i>cirh1a</i> mRNA, showing widespread expression at 20 hpf (A), followed by expression in the developing liver, gallbladder/pancreas, and anterior intestine at 3 dpf (B-C). (<b>D</b>) Close-up image of panel (C). (<b>E</b>) Immunohistochemistry and fluorescent in situ hybridization in 4 dpf <i>Tg</i>(bglob:EGFP) larvae, showing widespread <i>cirh1a</i> liver expression with focal expression in GFP-positive biliary cells (arrowheads). (<b>F</b>) Quantitation of <i>cirh1a</i> expression in GFP-positive biliary cells in 3 dpf and 4 dpf <i>Tg</i>(bglob:EGFP) larvae.</p
Activation of p53 pathway in Cirhin-deficient larvae.
<p>(A) RT-PCR with primers directed at cirh1a exons 13-15, demonstrating altered mRNA splicing following injection of IE14 MO (arrowhead). (B) Schematic of cirh1a alternative splicing due to IE14 MO, with alternate inclusion of intron 14 creating an in-frame stop codon. (C-E) Quantitative RT-PCR for tp53 (C) and p53 targets p21 (D) and mdm2 (E), expressed as fold increase over control mismatch MO-injected larvae. *, p<0.05 vs Mismatch MO 1 ng.</p
Biliary defects in Cirhin-deficient larvae are abrogated by <i>tp53</i> mutation.
<p>(A) Brightfield (left column) and fluorescent (right column) images of representative 5 dpf wild-type Cirhin-deficient larvae (BODIPY-FL C16 assay). Arrows indicate position of gallbladder. (B) Brightfield (left column) and fluorescent (right column) images of representative 5 dpf <i>tp53</i>-mutant Cirhin-deficient larvae (BODIPY-FL C16 assay). Arrows indicate position of gallbladder. (<b>C</b>) Quantitation of gallbladder fluorescence in Cirhin-deficient larvae. *, p<0.05 vs Tu mismatch MO-injected 1 ng; #, p<0.05 vs Tu <i>cirh1a</i> IE14 MO-injected 1 ng.</p
Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function.
<p>(<b>A</b>) Brightfield (left) and fluorescent (right) images of two <i>cirh1a</i> IE14 MO-injected 5 dpf larvae, 2 hours following their ingestion of BODIPY-FL C16 and fluorescent microspheres. Lower larva shows fluorescent lipid accumulation in gallbladder (single arrow and lower inset) indicating normal biliary function, while the other larva has defective biliary function and non-visible gallbladder (double arrow and upper inset), despite normal swallowing of substrate in both fish (fluorescent microspheres in gut, arrowheads). (<b>B</b>) Quantitation of gallbladder fluorescence in control and Cirhin-deficient larvae that had been injected with either the translation-blocking (ATG) or splice-blocking (IE14) MO. *, p<0.05 vs Mismatch MO 1 ng; #, p<0.05 vs Mismatch MO 1.5 ng; **, p<0.05 vs <i>cirh1a</i> ATG MO 0.5 ng; ***, p<0.05 vs <i>cirh1a</i> IE14 MO 0.5 ng.</p
Identification of the zebrafish Cirhin homolog.
<p>(<b>A</b>) Protein sequence alignments of human CIRHIN and zebrafish Cirhin. Identical residues are shaded black, and similar residues are shaded grey. Zebrafish Cirhin contains 685 amino acids, and is 54% identical and 72% similar to the human protein, with identity at the arginine residue mutated in NAIC (red arrowheads). (<b>B</b>) Synteny analyses of zebrafish chromosome 18 and human chromosome 16 near the <i>cirh1a</i> and <i>CIRH1A</i> loci. Diagonal lines indicate conserved syntenic relationships.</p
Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE) - Fig 2
<p><b>Calcium induces terminal differentiation of esophageal epithelial cells in-vitro A)</b> Cytokeratin 13 (CK13) and, <b>B)</b> involucrin (IVL), mRNA expression in response to high calcium (1.8mM CaCl<sub>2</sub>) media at 0, 24, 48, and 72 hours. Results are representative of three separate experiments. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 as compared to undifferentiated (0 hr) cells. Immunoblot for <b>C)</b> involucrin in calcium differentiated EPC2-hTERT cells. <b>D</b>) IHC for TSLP on organotypic cultures of esophageal epithelium. SSE = Stratified squamous epithelium; B = Basal epithelium, Sub = subepithelium.</p
Patient demographics, and allergy test results.
<p>Patient demographics, and allergy test results.</p
Terminal differentiation of human esophageal epithelial cells enhances the inducible expression and secretion of TSLP protein <i>in vitro</i>.
<p>TSLP mRNA expression in poly (I:C)-stimulated undifferentiated (<b>A</b>) and differentiated (<b>B</b>) EPC2-hTERT cells. <b>C)</b> TSLP protein secretion (pg/mL) by poly (I:C)-stimulated undifferentiated and differentiated EPC2-hTERT cells. <b>D</b>) TLR3 mRNA expression in undifferentiated and differentiated EPC2-hTERT cells. Results are representative of three separate experiments. * p<0.05, p<0.01***p <0.001, p<0.0001 as compared to undifferentiated cells.</p