Cox B.,Hospital for Sick Children |
Evangelou A.I.,Ontario Cancer Institute |
Whiteley K.,Samuel Lunenfeld Research Institute |
Ignatchenko V.,Ontario Cancer Institute |
And 9 more authors.
Molecular and Cellular Proteomics | Year: 2011
Preeclampsia (PE) adversely impacts ∼5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
PubMed | Aberystwyth University, Hospital for Sick Children, Toronto Center for Phenogenomics, University of Manitoba and 11 more.
Type: Journal Article | Journal: Nature genetics | Year: 2015
The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
Coultas L.,Hospital for Sick Children Research Institute |
Nieuwenhuis E.,Hospital for Sick Children Research Institute |
Anderson G.A.,Hospital for Sick Children |
Anderson G.A.,University of Toronto |
And 9 more authors.
Blood | Year: 2010
Despite the clear importance of Hedgehog (Hh) signaling in blood vascular development as shown by genetic analysis, its mechanism of action is still uncertain. To better understand the role of Hh in vascular development, we further characterized its roles in vascular development in mouse embryos and examined its interaction with vascular endothelial growth factor (VEGF), a well-known signaling pathway essential to blood vascular development. We found that VEGF expression in the mouse embryo depended on Hh signaling, and by using genetic rescue approaches, we demonstrated that the role of Hh both in endothelial tube formation and Notch-dependent arterial identity was solely dependent on its regulation of VEGF. In contrast, overactivation of the Hh pathway through deletion of Patched1 (Ptch1), a negative regulator of Hh signaling, resulted in reduced vascular density and increased Delta-like ligand 4 expression. The Ptch1 phenotype was independent of VEGF pathway dysregulation and was not rescued when Delta-like ligand 4 levels were restored to normal. These findings establish that Hh uses both VEGF- and Notch-dependent and -independent mechanisms to pattern specific events in early blood vascular development. © 2010 by The American Society of Hematology.
Cox B.J.,Hospital for Sick Children Research Institute |
Vollmer M.,Helmholtz Center for Environmental Research |
Tamplin O.,Hospital for Sick Children Research Institute |
Tamplin O.,University of Toronto |
And 13 more authors.
Genome Research | Year: 2010
Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, ∼10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/). © 2010 by Cold Spring Harbor Laboratory Press.
Lee W.,Hospital for Sick Children |
Donner E.J.,University of Toronto |
Nossin-Manor R.,Hospital for Sick Children |
Whyte H.E.A.,Hospital for Sick Children |
And 3 more authors.
Developmental Medicine and Child Neurology | Year: 2012
Aim The aim of this study was to determine the feasibility of undertaking visual functional magnetic resonance imaging (fMRI) in very preterm children. Method Forty-seven infants born at less than 32weeks gestational age (25 males, 22 females; mean (SD) age at birth 28.8wks [1.9]) were scanned using 1.5T MRI as part of a longitudinal neuroimaging study. These infants were scanned at preterm age (within 2wks of birth) and at term-equivalent age. Quantitative T2* data and fMRI in response to visual stimuli (flashing strobe) were acquired in this population. T2* values were compared at preterm age and at term-equivalent age using a two-tailed t-test. A general linear model was used to evaluate occipital lobe response to visual stimuli. Results T2* values were significantly higher at preterm age than at term-equivalent age in both the medial and lateral occipital lobes (preterm infants: 187.2ms and 198.4ms respectively; term infants: 110.9ms and 133.2ms respectively; p<0.002). Significant positive occipital lobe activation (q<0.01) was found in 3 out of 65 (5%) fMRIs carried out at preterm age and in 19 out of 26 (73%) scans carried out at term-equivalent age. Interpretation Visual stimuli do not elicit a reliable blood oxygen level-dependent (BOLD) response in very preterm infants during the preterm period. This suggests that BOLD fMRI may not be the appropriate modality for investigating occipital lobe function in very preterm infants. This article is commented on by Arichi on page of this issue © The Authors. Developmental Medicine & Child Neurology © 2012 Mac Keith Press.
Fujii H.,Hospital for Sick Children |
Luo Z.-J.,Hospital for Sick Children |
Kim H.J.,Hospital for Sick Children |
Newbigging S.,Toronto Center for Phenogenomics |
And 3 more authors.
PLoS ONE | Year: 2015
Chronic graft-versus-host disease (cGvHD) is the major source of late phase morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Humanized acute GvHD (aGvHD) in vivo models using NOD-SCID il2rγ-/- (NSG) mice are well described and are important tools for investigating pathogenicity of human cells in vivo. However, there have been only few reported humanized cGvHD mouse models. We evaluated if prolonged inflammation driven by low dose G-CSF-mobilized human PBMCs (G-hPBMCs) would lead to cGvHD following cyclophosphamide (CTX) administration and total body irradiation (TBI) in NSG mice. Engraftment was assessed in peripheral blood (PB) and in specific target organs by either flow cytometry or immunohistochemistry (IHC). Tissue samples were harvested 56 days post transplantation and were evaluated by a pathologist. Some mice were kept for up to 84 days to evaluate the degree of fibrosis. Mice that received CTX at 20mg/kg did not show aGvHD with stable expansion of human CD45+ CD3+ T-cells in PB (mean; 5.8 to 23.2%). The pathology and fibrosis scores in the lung and the liver were significantly increased with aggregation of T-cells and hCD68+ macrophages. There was a correlation between liver pathology score and the percentage of hCD68+ cells, suggesting the role of macrophage in fibrogenesis in NSG mice. In order to study long-term survival, 6/9 mice who survived more than 56 days showed increased fibrosis in the lung and liver at the endpoint, which suggests the infiltrating hCD68+ macrophages may be pathogenic. It was shown that the combination of CTX and TBI with a low number of G-hPBMCs (1×106) leads to chronic lung and liver inflammation driven by a high infiltration of human macrophage and mature human T cells from the graft, resulting in fibrosis of lung and liver in NSG mice. In conclusion this model may serve as an important pre-clinical model to further current understanding of the roles of human macrophages in cGvHD. © 2015 Fujii et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Hughes M.R.,University of British Columbia |
Anderson N.,University of Toronto |
Maltby S.,University of British Columbia |
Wong J.,University of British Columbia |
And 20 more authors.
Experimental Hematology | Year: 2011
Objective: Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS. Materials and Methods: We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1E924X). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1E924X mice at the biochemical, cellular, and pathophysiological levels. Results: Although Ank1E924X/E924X red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1E924X heterozygous mice, including a band similar in size to full-length ankyrin-1. Conclusions: The Ank1E924X strain provides a novel tool to study Ank1 and model HS. © 2011 ISEH - Society for Hematology and Stem Cells.
Traister A.,Hospital for Sick Children |
Walsh M.,Hospital for Sick Children |
Aafaqi S.,Hospital for Sick Children |
Lu M.,Hospital for Sick Children |
And 7 more authors.
PLoS ONE | Year: 2013
Rationale: Integrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype. However, ILK has been implicated as both a cardioprotective and oncogenic target, which imposes therapeutic constraints that are generally relevant to the translational potential of many kinases. Objective: To study the cardioprotective properties of the activation-resistant, non-oncogenic, mutation of ILK (ILK R211A) against experimental MI in vivo and Doxorubicin induced apoptosis in vitro and it's relationships to stress induced heat shock proteins. Methods/Results: The transgenic mouse heart over-expressing a point mutation in the ILK pleckstrin homology (PH) domain (TgR211A) exhibits a highly cardioprotective phenotype based on LAD-ligation-induced MI reduction in vivo, and on protection against doxorubicin (DOX)-induced cardiomyocyte apoptosis when overexpressed in human induced pluripotent stem cell (iPS)-derived cardiomyocytes in vitro. Intriguingly, the degree of cardioprotection seen with the ILKR211A mutation exceeded that with the ILKS343D mutation. Microarray and immunoprecipitation analyses revealed upregulation of expression levels and specific binding of ILKWT, ILKS343D and ILKR211A to both constitutively active heat-shock protein 70 (Hsc70) and inducible Hsp70 in response to MI, and to acute ILK overexpression in iPSC-cardiomyocytes. ILK-mediated cardioprotection was shown to depend upon Hsp70 ATPase activity. Conclusions: These findings indicate that wild type ILK and the non-oncogenic ILKR211A mutation comprise a cardioprotective module with Hsp/c70. These results advance a novel target discovery theme in which kinase mutations can be safely engineered to enhance cardioprotective effects. © 2013 Traister et al.