Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fi broblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1 ), NANOG , SOX2 , and NODAL , but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specifi c JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals. Introduction Mouse embryonic stem cells (mESC), conventionally derived from the inner cell mass (ICM) of preimplantation blastocysts, are pluripotent and can self-renew indefinitely in culture. mESC depend on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) to maintain the undifferentiated state [ 1 , 2 ]. Human embryonic stem cells (hESC) are also derived from blastocysts; however, these cells depend on basic fi broblast growth factor (bFGF) and Activin A for pluripotency and selfrenewal [ 3 , 4 ]. Interestingly, pluripotent cells derived from mouse epiblasts, a part of the ICM, also require bFGF and Activin A for pluripotency and self-renewal [ 5 , 6 ]. hESC share multiple features with mouse epiblast stem cells (mEpiSC), such as growing as fl at and compact colonies [5][6][7], they respond to BMP-4 by differentiating to trophectoderm (TE) [ 8 ] and germ cell progenitors [ 9 ], and they do not require LIF/ JAK/STAT3 signaling for self-renewal [ 4 , 10 ]. The functional and phenotypic similarities between these cell types suggest a developmental relationship [ 5 , 6 ], and demonstrate that there are at least 2 signaling mechanisms involved in capturing pluripotency in vitro. Recent reports show that bFGF and Activin A are also necessary for maintaining rabbit ESC [ 11 , 12 ], indicating that this signaling pathway may be a conserved mechanism of pluripotency in mammals.Here we tested this hypothesis in pigs, a representative of ungulates. Attempts to derive stem cells from pig embryos have traditionally reli...

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA+ background.

BackgroundEpigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.ResultsOur results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.ConclusionsOur study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.

BackgroundThe genome of the gastric pathogen Helicobacter pylori is characterised by considerable variation of both gene sequence and content, much of which is contained within three large genomic islands comprising the cag pathogenicity island (cagPAI) and two mobile integrative and conjugative elements (ICEs) termed tfs3 and tfs4. All three islands are implicated as virulence factors, although whereas the cagPAI is well characterised, understanding of how the tfs elements influence H. pylori interactions with different human hosts is significantly confounded by limited definition of their distribution, diversity and structural representation in the global H. pylori population.ResultsTo gain a global perspective of tfs ICE population dynamics we established a bioinformatics workflow to extract and precisely define the full tfs pan-gene content contained within a global collection of 221 draft and complete H. pylori genome sequences. Complete (ca. 35-55kbp) and remnant tfs ICE clusters were reconstructed from a dataset comprising > 12,000 genes, from which orthologous gene complements and distinct alleles descriptive of different tfs ICE types were defined and classified in comparative analyses. The genetic variation within defined ICE modular segments was subsequently used to provide a complete description of tfs ICE diversity and a comprehensive assessment of their phylogeographic context. Our further examination of the apparent ICE modular types identified an ancient and complex history of ICE residence, mobility and interaction within particular H. pylori phylogeographic lineages and further, provided evidence of both contemporary inter-lineage and inter-species ICE transfer and displacement.ConclusionsOur collective results establish a clear view of tfs ICE diversity and phylogeographic representation in the global H. pylori population, and provide a robust contextual framework for elucidating the functional role of the tfs ICEs particularly as it relates to the risk of gastric disease associated with different tfs ICE genotypes.Electronic supplementary materialThe online version of this article (10.1186/s13100-018-0109-4) contains supplementary material, which is available to authorized users.

Two distinct type IV secretion systems (T4SSs) can be identified in certain Helicobacter pylori strains, encoded on mobile genetic elements termed tfs3 and tfs4. Although their function remains unknown, both have been implicated in clinical outcomes of H. pylori infection. Here we provide evidence that the Tfs3 T4SS is required for activity of the pro-inflammatory Ser/Thr kinase protein, CtkA, in a gastric epithelial cell infection model. Previously, purified recombinant CtkA protein has been shown to upregulate NF-kappaB signalling and induce TNF-alpha and IL-8 cytokine secretion from cultured macrophages suggesting that it may potentiate the H. pylori-mediated inflammatory response. In this study, we show that CtkA expressed from its native host, H. pylori has a similar capacity for stimulation of a pro-inflammatory response from gastric epithelial cells. CtkA interaction was found to be dependent upon a complement of tfs3 T4SS genes, but independent of the T4SSs encoded by either tfs4 or the cag pathogenicity island. Moreover, the availability of CtkA for host cell interaction was shown to be conditional upon the carboxyl-terminus of CtkA, encoding a putative conserved secretion signal common to other variably encoded Tfs3 proteins. Collectively, our observations indicate a role for the Tfs3 T4SS in CtkA-mediated pro-inflammatory signalling by H. pylori and identify CtkA as a likely Tfs3 T4SS secretion substrate.

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Glioblastoma therapeutic challenges are in considerable part due to myriad survival adaptations and mechanisms, which allow malignant cells to repurpose signalling pathways within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space or promote evasion from repressive factors that represent anti-cancer defence mechanisms. We hypothesised that pre-clinical modelling of glioma invasion by recapitulating early events occurring immediately after surgery at the glioblastoma invasive margin, could reveal the cross-talk between malignant cells and the surrounding healthy astrocytes, which facilitates tumour recurrence. We first generated transgenic H1-derived neural stem cells using CRISPR/Cas9-mediated knock-in of the YFP reporter gene under the control of the GFAP promoter. Reproducible ultrahigh-throughput AggreWells™ (19,200 micro-wells per 24-well plate) were used to create astrocyte-glioblastoma organoids, which we term ‘Gliomasphere Matrices’. YFP-labelled astrocytes were co-cultured with 10 treatment-naïve patient-derived cell lines isolated from the 5-aminolevulinic (5ALA)-determined glioblastoma invasive margin. Co-cultures were seeded upon on a sequentially constructed, time-of-flight secondary ion mass spectrometry (ToF-SIMS)-characterised 3D scaffold, composed of decellularised human brain extract with defined PEGDA hydrogel. YFP-astrocytes were purified from each of the 10 Gliomasphere Matrices using fluorescence-activated cell sorting (FACS) after 6- and 10-days co-culture. RNAseq profiling to address both putative astrocytic reprogramming by invasive glioblastoma cells and gene expression changes intrinsic to tumour cells will be discussed in relation to RNAseq data from patient-derived 5ALA FACS-purified glioblastoma invasive margin tissue. This novel multi-faceted model offers a unique opportunity to recapitulate early molecular cross-talk which facilitates glioblastoma recurrence and may be utilised for high-throughput drug screening.

Glioblastoma therapeutic challenges are in considerable part due to myriad survival mechanisms which allow malignant cells to repurpose signalling pathways within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space. We hypothesised that pre-clinical modelling of glioma invasion by recapitulating early events occurring immediately after surgery at the glioblastoma invasive margin, could reveal the cross-talk between malignant cells and surrounding healthy astrocytes. We first generated transgenic H1-derived neural stem cells using CRISPR/Cas9-mediated knock-in of the YFP reporter gene under the control of the GFAP promoter at the AAVS1 safe harbour locus. Reproducible ultrahigh-throughput AggreWells™ (7200 mini-wells per plate) were used to create astrocyte-glioblastoma organoids, which we term ‘Gliomasphere Matrices’. YFP-labelled astrocytes were co-cultured with 10 treatment-naïve patient-derived cell lines isolated from the 5-aminolevulinic (5ALA)-determined glioblastoma invasive margin. Co-cultures were seeded upon a sequentially constructed, time-of-flight secondary ion mass spectrometry (ToF-SIMS)-characterised decellularised human brain extract. YFP-astrocytes were purified from each of the 10 Gliomasphere Matrices using fluorescence-activated cell sorting (FACS) after 6- and 10-days co-culture. RNA-sequencing of the putatively reprogrammed YFP-astrocytes showed the characteristic expression of canonical key regulators of multiple malignant diseases including high-grade glioma such as SND1 and EFNB2 in addition to the identification of a single novel marker located at chromosome 1 (C1orf61), highly expressed in malignant glioma when compared to somatic cancers according to TCGA RNA-sequencing data. Differentiated YFP-astrocytes also overexpressed IFITM2 and IFITM10, known to be involved in priming resistance against pathogenic microorganisms. This ultimately suggests a fluctuating state between malignant transformation imposed by the highly infiltrative glioma cells and the counter-action of the normal astrocytes to these deleterious invasive cells. This multi-faceted model offers a unique opportunity to recapitulate early molecular cross-talk which facilitates glioblastoma recurrence and may be utilised for high-throughput drug screening.