Now showing 1 - 10 of 13
  • Publication
    Wnt signalling is a bi-directional vulnerability of cancer cells
    Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.
      451Scopus© Citations 27
  • Publication
    Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega
    Multiple sequence alignments are fundamental to many sequence analysis methods. Most alignments are computed using the progressive alignment heuristic. These methods are starting to become a bottleneck in some analysis pipelines when faced with data sets of the size of many thousands of sequences. Some methods allow computation of larger data sets while sacrificing quality, and others produce high-quality alignments, but scale badly with the number of sequences. In this paper, we describe a new program called Clustal Omega, which can align virtually any number of protein sequences quickly and that delivers accurate alignments. The accuracy of the package on smaller test cases is similar to that of the high-quality aligners. On larger data sets, Clustal Omega outperforms other packages in terms of execution time and quality. Clustal Omega also has powerful features for adding sequences to and exploiting information in existing alignments, making use of the vast amount of precomputed information in public databases like Pfam.
      729Scopus© Citations 10532
  • Publication
    A Complete Analysis of HA and NA Genes of Influenza A Viruses
    Background: More and more nucleotide sequences of type A influenza virus are available in public databases. Although these sequences have been the focus of many molecular epidemiological and phylogenetic analyses, most studies only deal with a few representative sequences. In this paper, we present a complete analysis of all Haemagglutinin (HA) and Neuraminidase (NA) gene sequences available to allow large scale analyses of the evolution and epidemiology of type A influenza. Methodology/Principal Findings: This paper describes an analysis and complete classification of all HA and NA gene sequences available in public databases using multivariate and phylogenetic methods.Conclusions/Significance: We analyzed 18975 HA sequences and divided them into 280 subgroups according to multivariate and phylogenetic analyses. Similarly, we divided 11362 NA sequences into 202 subgroups. Compared to previous analyses, this work is more detailed and comprehensive, especially for the bigger datasets. Therefore, it can be used to show the full and complex phylogenetic diversity and provides a framework for studying the molecular evolution and epidemiology of type A influenza virus. For more than 85% of type A influenza HA and NA sequences into GenBank, they are categorized in one unambiguous and unique group. Therefore, our results are a kind of genetic and phylogenetic annotation for influenza HA and NA sequences. In addition, sequences of swine influenza viruses come from 56 HA and 45 NA subgroups. Most of these subgroups also include viruses from other hosts indicating cross species transmission of the viruses between pigs and other hosts. Furthermore, the phylogenetic diversity of swine influenza viruses from Eurasia is greater than that of North American strains and both of them are becoming more diverse. Apart from viruses from human, pigs, birds and horses, viruses from other species show very low phylogenetic diversity. This might indicate that viruses have not become established in these species. Based on current evidence, there is no simple pattern of inter-hemisphere transmission of avian influenza viruses and it appears to happen sporadically. However, for H6 subtype avian influenza viruses, such transmissions might have happened very frequently and multiple and bidirectional transmission events might exist.
      458Scopus© Citations 35
  • Publication
    Instability in progressive multiple sequence alignment algorithms
    Background: Progressive alignment is the standard approach used to align large numbers of sequences. As with all heuristics, this involves a trade off between alignment accuracy and computation time. Results: We examine this trade off and find that, because of a loss of information in the early steps of the approach, the alignments generated by the most common multiple sequence alignment programs are inherently unstable, and simply reversing the order of the sequences in the input file will cause a different alignment to be generated. Although this effect is more obvious with larger numbers of sequences, it can also be seen with data sets in the order of one hundred sequences. We also outline the means to determine the number of sequences in a data set beyond which the probability of instability will become more pronounced. Conclusions: This has major ramifications for both the designers of large-scale multiple sequence alignment algorithms, and for the users of these alignments.
      258Scopus© Citations 20
  • Publication
    GSK3 inhibitors regulate MYCN mRNA levels and reduce neuroblastoma cell viability through multiple mechanisms including p53 and Wnt signalling
    Neuroblastoma is an embryonal tumor accounting for approximately 15% of childhood cancer deaths. There exists a clinical need to identify novel therapeutic targets, particularly for treatment-resistant forms of neuroblastoma. Therefore, we investigated the role of the neuronal master regulator GSK3 in controlling neuroblastoma cell fate. We identified novel GSK3-mediated regulation of MYC (c-MYC and MYCN) mRNA levels, which may have implications for numerous MYC-driven cancers. In addition, we showed that certain GSK3 inhibitors induced large-scale cell death in neuroblastoma cells, primarily through activating apoptosis. mRNA-seq of GSK3 inhibitor–treated cells was performed and subsequent pathway analysis revealed that multiple signaling pathways contributed to the loss of neuroblastoma cell viability. The contribution of two of the signaling pathways highlighted by the mRNA-seq analysis was functionally validated. Inhibition of the p53 tumor suppressor partly rescued the cell death phenotype, whereas activation of canonical Wnt signaling contributed to the loss of viability, in a p53-independent manner. Two GSK3 inhibitors (BIO-acetoxime and LiCl) and one small-molecule Wnt agonist (Wnt Agonist 1) demonstrated therapeutic potential for neuroblastoma treatment. These inhibitors reduced the viability of numerous neuroblastoma cell lines, even those derived from high-risk MYCN-amplified metastatic tumors, for which effective therapeutics are currently lacking. Furthermore, although LiCl was lethal to neuroblastoma cells, it did not reduce the viability of differentiated neurons. Taken together our data suggest that these small molecules may hold potential as effective therapeutic agents for the treatment of neuroblastoma and other MYC-driven cancers.
      1945Scopus© Citations 69
  • Publication
    Making automated multiple alignments of very large numbers of protein sequences
    (Oxford University Press, 2013-02-21) ; ; ;
    Motivation: Recent developments in sequence alignment software have made possible multiple sequence alignments (MSAs) of >100 000 sequences in reasonable times. At present, there are no systematic analyses concerning the scalability of the alignment quality as the number of aligned sequences is increased. Results: We benchmarked a wide range of widely used MSA packages using a selection of protein families with some known structures and found that the accuracy of such alignments decreases markedly as the number of sequences grows. This is more or less true of all packages and protein families. The phenomenon is mostly due to the accumulation of alignment errors, rather than problems in guide-tree construction. This is partly alleviated by using iterative refinement or selectively adding sequences. The average accuracy of progressive methods by comparison with structure-based benchmarks can be improved by incorporating information derived from high-quality structural alignments of sequences with solved structures. This suggests that the availability of high quality curated alignments will have to complement algorithmic and/or software developments in the long-term.
      433Scopus© Citations 44
  • Publication
    Genes and signaling networks regulated during zebrafish optic vesicle morphogenesis
    Background: The genetic cascades underpinning vertebrate early eye morphogenesis are poorly understood. One gene family essential for eye morphogenesis encodes the retinal homeobox (Rx) transcription factors. Mutations in the human retinal homeobox gene (RAX) can lead to gross morphological phenotypes ranging from microphthalmia to anophthalmia. Zebrafish rx3 null mutants produce a similar striking eyeless phenotype with an associated expanded forebrain. Thus, we used zebrafish rx3-/- mutants as a model to uncover an Rx3-regulated gene network during early eye morphogenesis. Results: Rx3-regulated genes were identified using whole transcriptomic sequencing (RNA-seq) of rx3-/- mutants and morphologically wild-type siblings during optic vesicle morphogenesis. A gene co-expression network was then constructed for the Rx3-regulated genes, identifying gene cross-talk during early eye development. Genes highly connected in the network are hub genes, which tend to exhibit higher expression changes between rx3-/- mutants and normal phenotype siblings. Hub genes down-regulated in rx3-/- mutants encompass homeodomain transcription factors and mediators of retinoid-signaling, both associated with eye development and known human eye disorders. In contrast, genes up-regulated in rx3-/- mutants are centered on Wnt signaling pathways, associated with brain development and disorders. The temporal expression pattern of Rx3-regulated genes was further profiled during early development from maternal stage until visual function is fully mature. Rx3-regulated genes exhibited synchronized expression patterns, and a transition of gene expression during the early segmentation stage when Rx3 was highly expressed. Furthermore, most of these deregulated genes are enriched with multiple RAX-binding motif sequences on the gene promoter. Conclusions: Here, we assembled a comprehensive model of Rx3-regulated genes during early eye morphogenesis. Rx3 promotes optic vesicle morphogenesis and represses brain development through a highly correlated and modulated network, exhibiting repression of genes mediating Wnt signaling and concomitant enhanced expression of homeodomain transcription factors and retinoid-signaling genes.
      495Scopus© Citations 20
  • Publication
    Inhibition of the Pim1 Oncogene Results in Diminished Visual Function
    Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3-5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors results in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.
      356Scopus© Citations 20
  • Publication
    Systematic exploration of guide-tree topology effects for small protein alignments
    Background: Guide-trees are used as part of an essential heuristic to enable the calculation of multiple sequence alignments. They have been the focus of much method development but there has been little effort at determining systematically, which guide-trees, if any, give the best alignments. Some guide-tree construction schemes are based on pair-wise distances amongst unaligned sequences. Others try to emulate an underlying evolutionary tree and involve various iteration methods. Results: We explore all possible guide-trees for a set of protein alignments of up to eight sequences. We find that pairwise distance based default guide-trees sometimes outperform evolutionary guide-trees, as measured by structure derived reference alignments. However, default guide-trees fall way short of the optimum attainable scores. On average chained guide-trees perform better than balanced ones but are not better than default guide-trees for small alignments. Conclusions: Alignment methods that use Consistency or hidden Markov models to make alignments are less susceptible to sub-optimal guide-trees than simpler methods, that basically use conventional sequence alignment between profiles. The latter appear to be affected positively by evolutionary based guide-trees for difficult alignments and negatively for easy alignments. One phylogeny aware alignment program can strongly discriminate between good and bad guide-trees. The results for randomly chained guide-trees improve with the number of sequences.
      444Scopus© Citations 11
  • Publication
    OD-seq: outlier detection in multiple sequence alignments
    (BMC Informatics, 2015-08-25) ; ;
    Background: Multiple sequence alignments (MSA) are widely used in sequence analysis for a variety of tasks. Outlier sequences can make downstream analyses unreliable or make the alignments less accurate while they are being constructed. This paper describes a simple method for automatically detecting outliers and accompanying software called OD-seq. It is based on finding sequences whose average distance to the rest of the sequences in a dataset, is anomalous. Results: The software can take a MSA, distance matrix or set of unaligned sequences as input. Outlier sequences are found by examining the average distance of each sequence to the rest. Anomalous average distances are then found using the interquartile range of the distribution of average distances or by bootstrapping them. The complexity of any analysis of a distance matrix is normally at least O(N2 ) for N sequences. This is prohibitive for large N but is reduced here by using the mBed algorithm from Clustal Omega. This reduces the complexity to O(N log(N)) which makes even very large alignments easy to analyse on a single core. We tested the ability of OD-seq to detect outliers using artificial test cases of sequences from Pfam families, seeded with sequences from other Pfam families. Using a MSA as input, OD-seq is able to detect outliers with very high sensitivity and specificity. Conclusion: OD-seq is a practical and simple method to detect outliers in MSAs. It can also detect outliers in sets of unaligned sequences, but with reduced accuracy. For medium sized alignments, of a few thousand sequences, it can detect outliers in a few seconds.
      358Scopus© Citations 28