Biochemistry and Pharmacology - Research Publications
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ItemUnicycler: resolving bacterial genome assemblies from short and long sequencing reads( 2016-12-22)The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce more complete genome assemblies, but the sequencing is more expensive and error prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler utilises a novel semi-global aligner, which is used to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler .
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ItemAltered gp130 signalling ameliorates experimental colitis via myeloid cell-specific STAT3 activation and myeloid-derived suppressor cells(NATURE PORTFOLIO, 2016-02-05)STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130(757F/F) mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3(flox); gp130(757F/F) LysMcre/STAT3(flox)). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130(757F/F) mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines.
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ItemLoss of gastrokine-2 drives premalignant gastric inflammation and tumor progression(AMER SOC CLINICAL INVESTIGATION INC, 2016-04)Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.
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ItemThe MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome(BMJ PUBLISHING GROUP, 2016-07)OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1β and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1β, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.