Life Sciences Research for Lifelong Health

Publications bioinformatics

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Deficiency in spliceosome-associated factor CTNNBL1 does not affect ongoing cell cycling but delays exit from quiescence and results in embryonic lethality in mice.
Chandra A, van Maldegem F, Andrews S, Neuberger MS, Rada C

CTNNBL1 is an armadillo-repeat protein that associates with the CDC5L/Prp19 complex of the spliceosome. Unlike the majority of spliceosomal proteins (and despite having no obvious homologs), CTNNBL1 is inessential for cell viability as revealed by studies in both vertebrate B cell lines and in fission yeast. Here, however, we show that ablation of CTNNBL1 in the mouse germline results in mid-gestation embryonic lethality but that lineage-specific CTNNBL1 ablation in early B cell precursors does not affect the production and abundance of mature B lymphocytes. However, CTNNBL1-deficient resting B lymphocytes show sluggish exit from quiescence on cell activation, although once entry into cycle has initiated, proliferation and differentiation in response to mitogenic stimuli continue largely unaffected. A similar sluggish exit from quiescence is also observed on reprovision of nutrients to nitrogen-starved CTNNBL1-deficient yeast. The results indicate that, whereas other RNA splicing-associated factors have been connected to cell cycle progression, CTNNBL1 plays no essential role in cycling cells but does fulfill an evolutionarily conserved function in helping cells to undergo efficient exit from quiescence following activation.

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Cell cycle (Georgetown, Tex.), 12, 1551-4005, 732-42, 2013

PMID: 23343763

Open Access

Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing.
Kreck B, Richter J, Ammerpohl O, Barann M, Esser D, Petersen BS, Vater I, Murga Penas EM, Bormann Chung CA, Seisenberger S, Lee Boyd V, Smallwood S, Drexler HG, Macleod RA, Hummel M, Krueger F, Häsler R, Schreiber S, Rosenstiel P, Franke A, Siebert R

Leukemia, 27, 1476-5551, 1751-3, 2013

PMID: 23307032

Open Access

DNA methylation profiles define stem cell identity and reveal a tight embryonic-extraembryonic lineage boundary.
CE Senner, F Krueger, D Oxley, S Andrews, M Hemberger

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.

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Stem cells (Dayton, Ohio), 30, 12, 2732-45, 2012

PMID: 23034951
DOI: 10.1002/stem.1249

Open Access

Large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling.
F Krueger, SR Andrews, CS Osborne

Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.

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PloS one, 6, 1, e16607, 2011

PMID: 21305042
DOI: 10.1371/journal.pone.0016607

Meet the neighbours: tools to dissect nuclear structure and function.
CS Osborne, PA Ewels, AN Young

The eukaryotic cell nucleus displays a high degree of spatial organization, with discrete functional subcompartments that provide microenvironments where specialized processes take place. Concordantly, the genome also adopts defined conformations that, in part, enable specific genomic regions to interface with these functional centers. Yet the roles of many subcompartments and the genomic regions that contact them have not been explored fully. More fundamentally, it is not entirely clear how genome organization impacts function, and vice versa. The past decade has witnessed the development of a new breed of methods that are capable of assessing the spatial organization of the genome. These stand to further our understanding of the relationship between genome structure and function, and potentially assign function to various nuclear subcompartments. Here, we review the principal techniques used for analyzing genomic interactions, the functional insights they have afforded and discuss the outlook for future advances in nuclear structure and function dynamics.

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Briefings in functional genomics, 10, 1, 11-7, 2011

PMID: 21258046
DOI: 10.1093/bfgp/elq034

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes.
S Tomizawa, H Kobayashi, T Watanabe, S Andrews, K Hata, G Kelsey, H Sasaki

Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.

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Development (Cambridge, England), 138, 5, 811-20, 2011

PMID: 21247965
DOI: 10.1242/dev.061416

Open Access

PI3K signaling through the dual GTPase-activating protein ARAP3 is essential for developmental angiogenesis.
L Gambardella, M Hemberger, B Hughes, E Zudaire, S Andrews, S Vermeren

One function of phosphoinositide 3-kinase α (PI3Kα), which generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], is its regulation of angiogenesis in the developing embryo and in pathological situations. ARAP3 is a PtdIns(3,4,5)P(3)- and Rap-activated guanosine triphosphatase (GTPase)-activating protein (GAP) for the small GTPases RhoA and Arf6. Here, we show that deleting Arap3 in the mouse caused embryonic death in mid-gestation due to an endothelial cell-autonomous defect in sprouting angiogenesis. Explants taken at a developmental stage at which no defect was yet present reproduced this phenotype ex vivo, demonstrating that the defect was not secondary to hypoxia, placental defects, or organ failure. In addition, knock-in mice expressing an ARAP3 point mutant that cannot be activated by PtdIns(3,4,5)P(3) had angiogenesis defects similar to those of Arap3(-/-) embryos. Our work delineates a previously unknown signaling pathway that controls angiogenesis immediately downstream of PI3Kα through ARAP3 to the Rho and Arf family of small GTPases.

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Science signaling, 3, 145, ra76, 2010

PMID: 20978237
DOI: 10.1126/scisignal.2001026

Open Access

What is the advantage of a transient precursor in autophagosome biogenesis?
NT Ktistakis, S Andrews, J Long

We have recently proposed that some autophagosomes are formed within omegasomes, membrane sites connected to the endoplasmic reticulum and enriched in phosphatidylinositol 3-phosphate. In order to understand if there is any biological advantage to having such a precursor in autophagosome biogenesis, we generated a simple computer program that simulates omegasome and autophagosome formation under a variety of conditions. We concluded from running this simulation that having a transient precursor permits a bigger dynamic range of the autophagic response and allows a more efficient approach to steady state after autophagy stimulation.

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Autophagy, 7, 1, 118-22, 2011

PMID: 20935487
DOI: 10.1083/jcb.200803137

Open Access

Difference Tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters.
S Andrews, J Gilley, MP Coleman

Studies of axonal transport are critical, not only to understand its normal regulation, but also to determine the roles of transport impairment in disease. Exciting new resources have recently become available allowing live imaging of axonal transport in physiologically relevant settings, such as mammalian nerves. Thus the effects of disease, ageing and therapies can now be assessed directly in nervous system tissue. However, these imaging studies present new challenges. Manual or semi-automated analysis of the range of transport parameters required for a suitably complete evaluation is very time-consuming and can be subjective due to the complexity of the particle movements in axons in ex vivo explants or in vivo. We have developed Difference Tracker, a program combining two new plugins for the ImageJ image-analysis freeware, to provide fast, fully automated and objective analysis of a number of relevant measures of trafficking of fluorescently labeled particles so that axonal transport in different situations can be easily compared. We confirm that Difference Tracker can accurately track moving particles in highly simplified, artificial simulations. It can also identify and track multiple motile fluorescently labeled mitochondria simultaneously in time-lapse image stacks from live imaging of tibial nerve axons, reporting values for a number of parameters that are comparable to those obtained through manual analysis of the same axons. Difference Tracker therefore represents a useful free resource for the comparative analysis of axonal transport under different conditions, and could potentially be used and developed further in many other studies requiring quantification of particle movements.

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Journal of neuroscience methods, 193, 2, 281-7, 2010

PMID: 20869987
DOI: 10.1016/j.jneumeth.2010.09.007

Deletion of the RNA-binding proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T lymphoblastic leukemia.
DJ Hodson, ML Janas, A Galloway, SE Bell, S Andrews, CM Li, R Pannell, CW Siebel, HR MacDonald, K De Keersmaecker, AA Ferrando, G Grutz, M Turner

ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.

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Nature immunology, 11, 8, 717-24, 2010

PMID: 20622884
DOI: 10.1038/ni.1901

Open Access

CD8 locus nuclear dynamics during thymocyte development.
E Ktistaki, A Garefalaki, A Williams, SR Andrews, DM Bell, KE Foster, CG Spilianakis, RA Flavell, N Kosyakova, V Trifonov, T Liehr, D Kioussis

Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.

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Journal of immunology (Baltimore, Md. : 1950), 184, 10, 5686-95, 2010

PMID: 20404270
DOI: 10.4049/jimmunol.1000170

Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency.
C Popp, W Dean, S Feng, SJ Cokus, S Andrews, M Pellegrini, SE Jacobsen, W Reik

Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases AID and APOBEC1 can deaminate 5-methylcytosine in vitro and in Escherichia coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulphite next generation sequencing in wild-type and AID-deficient mouse PGCs at embryonic day (E)13.5. Wild-type PGCs revealed marked genome-wide erasure of methylation to a level below that of methylation deficient (Np95(-/-), also called Uhrf1(-/-)) embryonic stem cells, with female PGCs being less methylated than male ones. By contrast, AID-deficient PGCs were up to three times more methylated than wild-type ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in AID-deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. AID deficiency interferes with genome-wide erasure of DNA methylation patterns, indicating that AID has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.

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Nature, 463, 7284, 1101-5, 2010

PMID: 20098412
DOI: 10.1038/nature08829

Open Access

Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.
S Schoenfelder, T Sexton, L Chakalova, NF Cope, A Horton, S Andrews, S Kurukuti, JA Mitchell, D Umlauf, DS Dimitrova, CH Eskiw, Y Luo, CL Wei, Y Ruan, JJ Bieker, P Fraser

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

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Nature genetics, 42, 1, 53-61, 2010

PMID: 20010836
DOI: 10.1038/ng.496

Open Access

ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences.
HE Craig, J Coadwell, H Guillou, S Vermeren

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P(3)) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P(3,) which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P(3) in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P(3) in isolation. Instead, the minimal PtdIns(3,4,5)P(3) binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P(3), however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P(3) binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P(3) binding.

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Cellular signalling, 22, 2, 257-64, 2010

PMID: 19786092
DOI: 10.1016/j.cellsig.2009.09.025

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.
LS Matheson, MJ Osborn, JA Smith, D Corcos, M Hamon, R Chaouaf, J Coadwell, G Morgan, D Oxley, M Brüggemann

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

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International immunology, 21, 8, 957-66, 2009

PMID: 19561045
DOI: 10.1093/intimm/dxp062

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors).
P Raghu, M Manifava, J Coadwell, NT Ktistakis

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.

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Biochimica et biophysica acta, 1791, 9, 889-97, 2009

PMID: 19345277
DOI: 10.1016/j.bbalip.2009.03.013

Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.
CR Farthing, G Ficz, RK Ng, CF Chan, S Andrews, W Dean, M Hemberger, W Reik

DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.

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PLoS genetics, 4, 6, e1000116, 2008

PMID: 18584034
DOI: 10.1371/journal.pgen.1000116

Open Access

The correlation determinant in tests for synchronization in neuronal spike data.
E Walters, A Segonds-Pichon, AU Nicol

We present a statistical approach to the identification of correlated activity in multineuron spike data, based on the value of the correlation determinant. This approach is not compromised by the lack of independence often encountered in this kind of data. We illustrate our method by applying it both to simulated data and to data recorded from neurons in a forebrain region (intermediate medial mesopallium, IMM) of the behaving domestic chick and simultaneously from the corresponding contralateral region. There is no direct anatomical connection between the two sites, and the validity of this technique is strongly supported by the observation that when the test indicates significantly correlated activity for neurons within either hemisphere, this correlation is greatly reduced, and ultimately obliterated, by serial incorporation of activity from neurons in the opposite hemisphere. Since the value of individual correlation coefficients allied to the Bonferroni correction is often used as a diagnostic tool, we also present comparisons of that approach with our correlation determinant approach.

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Journal of neuroscience methods, 172, 1, 60-6, 2008

PMID: 18499264
DOI: 10.1016/j.jneumeth.2008.04.003

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination.
S Donald, T Humby, I Fyfe, A Segonds-Pichon, SA Walker, SR Andrews, WJ Coadwell, P Emson, LS Wilkinson, HC Welch

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

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Proceedings of the National Academy of Sciences of the United States of America, 105, 11, 4483-8, 2008

PMID: 18334636
DOI: 10.1073/pnas.0712324105

Open Access

PI3K class IB pathway in neutrophils.
S Andrews, L Stephens, P Hawkins

Activation of G(i)-coupled receptors in neutrophils stimulates class IB phosphoinositide 3-kinase (PI3K) (also known as PI3Kgamma) through the combined actions of Gbetagamma subunits and the small guanosine triphosphatase (GTPase) Ras, resulting in the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in the plasma membrane. In most cases, the effectors of this pathway possess a pleckstrin homology (PH) domain that mediates the interaction with and regulation by these two lipid messengers. These direct effectors sit within a complex regulatory network that includes several other signaling pathways and that is responsible for the control of important neutrophil functions, including adhesion, chemotaxis, secretion, and the "respiratory burst" [activation of the nicotinamide adenosine diphosphate (NADPH) oxidase]. Although the molecular details that link the direct effectors of class IB PI3K to these complex cell responses are still largely unknown, these responses involve complex regulation of small GTPases of the Rac, Rho, and Arf families.

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Science's STKE : signal transduction knowledge environment, 2007, 407, cm3, 2007

PMID: 17925574
DOI: 10.1126/stke.4072007cm3

PI3K class IB pathway.
Andrews S, Stephens LR, Hawkins PT

Class I phosphoinositide 3-kinases (PI3Ks) are well-established signal transduction enzymes that play an important role in the mechanisms by which a wide variety of cell surface receptors control several cellular functions, including cellular growth, division, survival, and movement. Class IB PI3K (also known as PI3Kgamma) allows fast-acting, heterotrimeric GTP-binding protein-coupled receptors to access this pathway. Activation of class IB PI3K results in the rapid synthesis of phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and its dephosphorylation product, PI(3,4)P2, in the plasma membrane. These two lipid messengers bind to multiple, pleckstrin homology (PH) domain-containing effectors, which together regulate a complex signaling web downstream of receptor activation. This pathway regulates the activity of protein kinases and small guanosine triphosphatases that control cellular movement, adhesion, contraction, and secretion. Most of the ligands that have been established to activate class IB PI3K are involved in coordinating the body's response to injury and infection through the regulation of multiple cell types in the immune system and vascular lining. Mice lacking the catalytic subunit of class IB PI3K are remarkably resistant to the development of several inflammatory pathologies in mouse models of human inflammatory disease. These results suggest small molecule inhibitors of class IB PI3K may represent a novel class of therapeutic agents that may complement existing anti-inflammatory treatments.

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Science's STKE : signal transduction knowledge environment, 2007, 1525-8882, cm2, 2007

PMID: 17925573

Open Access

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis.
GJ Ferguson, L Milne, S Kulkarni, T Sasaki, S Walker, S Andrews, T Crabbe, P Finan, G Jones, S Jackson, M Camps, C Rommel, M Wymann, E Hirsch, P Hawkins, L Stephens

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

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Nature cell biology, 9, 1, 86-91, 2007

PMID: 17173040
DOI: 10.1038/ncb1517

P-Rex1 regulates neutrophil function.
Welch HC, Condliffe AM, Milne LJ, Ferguson GJ, Hill K, Webb LM, Okkenhaug K, Coadwell WJ, Andrews SR, Thelen M, Jones GE, Hawkins PT, Stephens LR

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.

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Current biology : CB, 15, 0960-9822, 1867-73, 2005

PMID: 16243035

Open Access