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Ablett M.P.,University of Manchester | O'Brien C.S.,University of Manchester | Sims A.H.,Institute of Genetics and Molecular Medicine | Farnie G.,University of Manchester | Clarke R.B.,University of Manchester
Oncotarget | Year: 2014

C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis resistant (AR) cells were collected from immortalised (MCF10A, 226L) and malignant (MCF7, T47D, SKBR3) breast cell lines and assessed for stem cell enrichment versus unsorted cells. AR cells had significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR normal cells demonstrated increased formation of 3D structures in Matrigel compared to unsorted cells. In vivo, SKBR3 and T47D AR cells had 7- and 130-fold enrichments for tumour formation respectively, compared with unsorted cells. AR cells contained significantly elevated CXCR4 transcript and protein levels compared to unsorted cells. Importantly, CXCR4 mRNA was higher in stem cellenriched CD44+/CD24- patient-derived breast cancer cells compared to non-enriched cells. CXCR4 stimulation by its ligand SDF-1 reduced MFE of the normal breast cells lines but increased the MFE in T47D and patient-derived breast cancer cells. CXCR4 inhibition by AMD3100 increased stem cell activity but reduced the selfrenewal capacity of the malignant breast cell line T47D. CXCR4+ FACS sorted MCF7 cells demonstrated a significantly increased MFE compared with CXCR4- cells. This significant increase in MFE was further demonstrated in CXCR4 over-expressing MCF7 cells which also had an increase in self-renewal compared to parental cells. A greater reduction in self-renewal following CXCR4 inhibition in the CXCR4 over-expressing cells compared with parental cells was also observed. Our data establish for the first time that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. Here, we demonstrate that CXCR4 signalling specifically regulates breast cancer stem cell activities and may therefore be important in tumour formation at the sites of metastases.

Wright A.F.,Institute of Genetics and Molecular Medicine
Nature Genetics | Year: 2011

A careful analysis of risk haplotypes in relation to age-related macular degeneration (AMD) susceptibility has led to the identification of a rare, high-penetrance variant in the complement factor H (CFH) gene that is also causally associated with atypical hemolytic uremic syndrome (aHUS) and related glomerulopathies. This finding provides a convincing causal mechanism linking the two diseases and develops a paradigm for the genetic architecture of a common and complex disease. © 2011 Nature America, Inc. All rights reserved.

Wright A.F.,Institute of Genetics and Molecular Medicine | Chakarova C.F.,University College London | Abd El-Aziz M.M.,University College London | Bhattacharya S.S.,University College London
Nature Reviews Genetics | Year: 2010

The retina provides exquisitely sensitive vision that relies on the integrity of a uniquely vulnerable cell, the photoreceptor (PR). The genetic and mechanistic causes of retinal degeneration due to PR cell death-which occurs in conditions such as retinitis pigmentosa and age-related macular degeneration-are being successfully dissected. Over one hundred loci, some containing common variants but most containing rare variants, are implicated in the genetic architecture of this complex trait. This genetic heterogeneity results in equally diverse disease mechanisms that affect almost every aspect of PR function but converge on a common cell death pathway. Although genetic and mechanistic diversity creates challenges for therapy, some approaches- particularly gene-replacement therapy-are showing considerable promise. © 2010 Macmillan Publishers Limited. All rights reserved.

O'Connell M.A.,Institute of Genetics and Molecular Medicine
Advances in Experimental Medicine and Biology | Year: 2010

From analysis of deep-sequencing data it is apparent that sequence differences occur between the genome and miRNAs. Changes from genomic A to an apparent G in miRNA can be accounted for by the editing activity of ADARs. Questions that arise from this observation are: How many miRNAs are edited and to what frequency? Is there a specific step in the biogenesis of miRNAs that is preferentially susceptible to editing by ADARs? However the key question is whether editing affects the downstream activity of miRNAs. Despite much evidence that miRNAs are edited, critical examination of the functional data shows a dearth of examples where editing has been demonstrated to actually affect the downstream miRNA activity in vivo. Even where it is demonstrated that RNA editing can affect biogenesis or targeting of a particular miRNA, effects may be limited by redundancy within the miRNA network. © 2010 Landes Bioscience and Springer Science+Business Media, LLC.

Overton I.M.,Institute of Genetics and Molecular Medicine | Barton G.J.,University of Dundee
Methods | Year: 2011

Selection of protein targets for study is central to structural biology and may be influenced by numerous factors. A key aim is to maximise returns for effort invested by identifying proteins with the balance of biophysical properties that are conducive to success at all stages (e.g. solubility, crystallisation) in the route towards a high resolution structural model. Selected targets can be optimised through construct design (e.g. to minimise protein disorder), switching to a homologous protein, and selection of experimental methodology (e.g. choice of expression system) to prime for efficient progress through the structural proteomics pipeline. Here we discuss computational techniques in target selection and optimisation, with more detailed focus on tools developed within the Scottish Structural Proteomics Facility (SSPF); namely XANNpred, ParCrys, OB-Score (target selection) and TarO (target optimisation). TarO runs a large number of algorithms, searching for homologues and annotating the pool of possible alternative targets. This pool of putative homologues is presented in a ranked, tabulated format and results are also visualised as an automatically generated and annotated multiple sequence alignment. The target selection algorithms each predict the propensity of a selected protein target to progress through the experimental stages leading to diffracting crystals. This single predictor approach has advantages for target selection, when compared with an approach using two or more predictors that each predict for success at a single experimental stage. The tools described here helped SSPF achieve a high (21%) success rate in progressing cloned targets to diffraction-quality crystals. © 2011 Elsevier Inc.

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