Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing.

Globally, hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Epidemiology and clinical presentation of hepatitis E vary greatly by location and are affected by the HEV genotype. Nucleic acid amplification technique (NAT)-based assays are important for the detection of acute HEV infection as well for monitoring chronic cases of hepatitis E. The aim of the study was to evaluate a panel of samples containing different genotypes of HEV for use in nucleic NAT-based assays. The panel of samples comprises eleven different members including HEV genotype 1a (2 strains), 1e, 2a, 3b, 3c, 3e, 3f, 4c, 4g as well as a human isolate related to rabbit HEV. Each laboratory assayed the panel members directly against the 1 World Health Organization (WHO) International Standard (IS) for HEV RNA (6329/10) which is based upon a genotype 3 a strain. The samples for evaluation were distributed to 24 laboratories from 14 different countries and assayed on three separate days. Of these, 23 participating laboratories returned a total of 32 sets of data; 17 from quantitative assays and 15 from qualitative assays. The assays used consisted of a mixture of in-house developed and commercially available assays. The results showed that all samples were detected consistently by the majority of participants, although in some cases, some samples were detected less efficiently. Based on the results of the collaborative study the panel (code number 8578/13) was established as the "1st International Reference Panel (IRP) for all HEV genotypes for NAT-based assays" by the WHO Expert Committee on Biological Standardization. This IRP will be important for assay validation and ensuring adequate detection of different genotypes and clinically important sub-genotypes of HEV

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P-2, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P-2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P-2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.Shared first authorship: Anja Nitzsche, Riikka Pietilä and Dominic T Love</p

Gpr116 Receptor Regulates Distinctive Functions in Pneumocytes and Vascular Endothelium.

Despite its known expression in both the vascular endothelium and the lung epithelium, until recently the physiological role of the adhesion receptor Gpr116/ADGRF5 has remained elusive. We generated a new mouse model of constitutive Gpr116 inactivation, with a large genetic deletion encompassing exon 4 to exon 21 of the Gpr116 gene. This model allowed us to confirm recent results defining Gpr116 as necessary regulator of surfactant homeostasis. The loss of Gpr116 provokes an early accumulation of surfactant in the lungs, followed by a massive infiltration of macrophages, and eventually progresses into an emphysema-like pathology. Further analysis of this knockout model revealed cerebral vascular leakage, beginning at around 1.5 months of age. Additionally, endothelial-specific deletion of Gpr116 resulted in a significant increase of the brain vascular leakage. Mice devoid of Gpr116 developed an anatomically normal and largely functional vascular network, surprisingly exhibited an attenuated pathological retinal vascular response in a model of oxygen-induced retinopathy. These data suggest that Gpr116 modulates endothelial properties, a previously unappreciated function despite the pan-vascular expression of this receptor. Our results support the key pulmonary function of Gpr116 and describe a new role in the central nervous system vasculature

Normalized pathological angiogenesis in <i>Gpr116</i>^-/- retinas.

<p>A. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (the images shown are representative of 5 mice per genotype). B. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype). C. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (n = 5 mice at least per genotype). D. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (n≥7 mice at least per genotype). E. Confocal images of post-OIR tufts (blue arrows) in <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype)</p

Retinal vascular patterning in <i>Gpr116</i>^-/- mice.

<p>A. Vascular network in P4 retinas. Dashed line indicates the limits of the retina (the picture shown is representative of at least 5 mice for each genotype). B. Quantification of the retinal vascular outgrowth at P4 (n = 5 for WT, n = 12 for heterozygotes and n = 6 for knockout). C. Vascular patterning in P7 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. Isolectin (red), CD31 (green) and Erg (grey) were used to visualize endothelium, and NG2 (green) and ASMA (red) to detect mural cells (the images shown are representative of 3 mice for each genotype). D. Vascular patterning in P7 retinas from <i>Gpr116</i> ECKO and littermates controls. Isolectin (red) is used to visualize endothelium, and NG2 (green) and smooth muscle actin α (ASMA, blue) to detect mural cells (the images show are representative of 2 mice per genotype). E. Isolectin (red) and FITC-dextran (green) distribution in P21 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. CD31 (green) is used to stain the endothelium, and nuclei are stained with Hoechst (blue) (the images shown are representative of 3 mice per genotype). F. Monolayers formed by isolated endothelial cells from <i>Gpr116</i> WT, heterozygous and knockout brain. Endothelial cells (CD31) and nuclei (Hoechst) are indicated in green and blue, respectively (the pictures shown are representative of 3 mice for each genotype)</p

Massive accumulation phenotype in lungs of aged <i>Gpr116</i>^-/- mice.

<p>A. Bright field image of the inflated lung from <i>Gpr116</i> WT, heterozygous and knockout littermates. B. Weights of whole lungs over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). C. Bright field images of heart from <i>Gpr116</i> WT, heterozygous and knockout littermates. D. Weights of the heart (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). E. Bright field images of the spleen from <i>Gpr116</i> WT, heterozygous and knockout littermates. F. Weights of the spleen (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). G. BALF collected from <i>Gpr116</i> WT, heterozygous and knockout littermates (The picture shown is representative of 3 mice for each genotype). H. Quantification of saturated phosphatydilcholine in BALF by ELISA (n = 3 mice per genotype). I. Quantification of protein content in BALF by BCA assay (n = 3 mice per genotype). J. Surfactant proteins detection in BALF by western blot. Molecular weights are indicated on the right. (n = 2 mice per genotype). K. Bright field images of the lung, after hematoxylin and eosin staining. The black arrowheads indicate alveolar macrophages (the image is representative of 4 mice for each genotype). L. Electron microscopy view of <i>Gpr116</i> wildtype and knockout lungs (n = 2 mice for each genotype). M. Confocal images of lung sections stained with ADRP (white) and nuclear stain (Hoechst, blue). Note that a red autofluorescent signal appears in knockout lungs. (the image shown is representative of 2 mice for each genotype). N. Confocal images of lung sections stained with nuclear marker Hoechst (blue) to show autofluorescent cells accumulated in the alveolar space, either in the green or red channel (the image is representative of 3 mice for each genotype). O. Autofluorescence emission spectrum of macrophages in the old knockout lung, upon 405 nm excitation (the image is representative of 2 mice). P. Detection of autofluorescent cells from <i>Gpr116</i> knockout lung by FACS (n = 2 mice per genotype)</p

Blood brain barrier breakdown in <i>Gpr116</i>^-/- mice.

<p>A. Whole brain images taken after 1kDa cadaverine perfusion (left) and associated quantification of extravasated cadaverine (right) in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n≥5 mice for each genotype). B. Whole brain images taken 70 kDa tetramethylrhodamine dextran perfusion (left) and quantification of extravasated tracer (right) in <i>Gpr116</i> WT and heterozygous and <i>Gpr116</i> ECKO mice (n = 3 for wild type and ECKO, n = 2 for <i>PDGF-B</i>^{<i>ret/ret</i>}, n = 1 for uninjected control). C. Confocal images of cerebral cortex from aged <i>Gpr116</i> WT, heterozygous and knockout mice. Astrocytes (GFAP) appear in green, endothelial cells (CD31) in red (the images are representative of 4 mice per genotype) and associated quantification of perivascular associated astrocytes in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n = 4 mice for each genotype, 2 sections at least quantified per genotype). D. Whole brain fluorescence images taken after Alexa 555-cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 1.5-month-old <i>Gpr116</i> knockout (n = 3 mice per genotype). E. Whole brain fluorescent images taken after cadaverine circulation (upper) and associated quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> AEC KO (n = 6 mice per genotype). F. Whole brain fluorescent images taken after cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> ECKO (n = 7 mice per genotype)</p

Vascular expression and genetic ablation of the <i>Gpr116</i> gene in mouse.

<p>A. <i>Gpr116</i> mRNA expression in the published organ-specific EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref045" target="_blank">45</a>]. B. <i>Gpr116</i> mRNA expression assessed by qRT-PCR in EC from 3-weeks-old and 3-months-old ROSA^mT/mG x Tie2-Cre mice. Results are normalized by brain EC expression. Error bars represent SD. (n = 3 mice per genotype). C. <i>Gpr116</i> mRNA expression in the published brain-specific vascular and EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref046" target="_blank">46</a>]. D. Schematic representation of the area targeted by homologous recombination in the <i>Gpr116</i> locus. Dotted lines indicate the regions of homology in between the Gpr116 locus and the cassette. The dark grey arrow indicates the position of WT primers: both are located in the untranslated region of exon 21, but the area recognized by the forward primer is lost in the mutant allele. The light grey arrow represents the knockout primer, specific for the cassette. Critical Gpr116 domains (SEA, IgG, GAIN and transmembraine, TM) are indicated above the corresponding encoding exons. E. Example of genotyping PCR products on genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. WT primers amplify a 325-bp fragment in the 3´UTR exon 21 of <i>Gpr116</i> gene representing the wild type allele. The 401 bp band is specific for the mutant allele. F. Example of genotyping PCR products using genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. LacZ primers amplify a 210 bp fragment in LacZ gene present in the insert replacing exon 4 to 21. G. <i>Gpr116</i> exon 17–18 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). H. <i>Gpr116</i> exon 2–3 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). I. mRNA detection by RNAscope in brain cortical capillary vessels from <i>Gpr116</i> WT (top row), knockout (middle row) and ROSA^mTmG X Tie2-Cre mice (lower row) at 3 weeks. On the left column, note that only the probe signal (red) and the nuclear staining (blue) are visible. On the right column, an endothelial staining (green) is merged to the probe and the nuclear signal: a CD31 antibody staining is on the two upper rows, while Tie-2 Cre mediated GFP is on the lower row. (n = 1 mouse per genotype).</p