Human liver cancer research currently lacks in vitro models that faithfully recapitulate the pathophysiology of the original tumour. We recently described a novel, near-physiological organoid culture system, where primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here, we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumours. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumour, allowing discrimination between different tumour tissues and subtypes, even after long term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumourogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug screening testing and lead to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized medicine approaches for the disease.

Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.DOI: http://dx.doi.org/10.7554/eLife.26575.001

Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scribKD) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scribKD cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.

The rapid, reductive early divisions of many metazoan embryos are followed by the mid-blastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the mid-blastula transition (MBT). We show that four DNA replication factors, Cut5, RecQ4, Treslin, and Drf1, are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis . The levels of these factors regulate multiple events of the MBT including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.The early cell divisions in many metazoa are rapid and maintained by maternally supplied products. Bulk zygotic transcription begins at the mid-blastula transition (MBT), also referred to as the start of the maternal-zygotic transition (MZT) (1), and is accompanied by the onset of cell movement, the activation of cell cycle checkpoints and the elongation and asynchrony of cell divisions (2-6). This elongation of the cell cycle is coincident with a reduction in the density and synchrony of DNA replication initiation events (7-9), leading to the possibility that replication factors themselves might be important regulators of cell cycle duration during development. To test this hypothesis we measured the abundance of replication initiation factors in Xenopus laevis embryos. Components of the first step of replication initiation, a process called 'licensing' or pre-replicative complex (pre-RC) assembly, remained constant or increased in abundance during embryogenesis ( After pre-RC formation, two kinases are required to assemble active replisomes, CyclinDependent Kinase (CDK) and Dbf4-Dependent kinase (DDK). The abundance of the noncatalytic subunit of DDK, which in Xenopus embryos is Drf1 (Dbf4-related factor 1), becomes greatly reduced by the time of gastrulation (10, 11). Both the mRNA and protein levels of three other replication factors, cut5, treslin and recq4 also decreased during embryogenesis ( Fig. 1A and Fig. S1). (12)(13)(14). Reduction in the levels of these replication factors during early cell divisions suggested that their abundance might be a determinant of embryonic replication initiation rates. To test this, we added in vitro translated (IVT) RecQ4, Treslin, Cut5, and Drf1 to Xenopus egg extracts and analyzed the replication of sperm nuclei in vitro. Increased expression of these proteins in extracts resulted in a corresponding increase in DNA synthesis when these factors were overexpressed in combination ( Fig. S2A-E). Furthermore, increasing the nuclei:extract ratio, to mimic the increasing N/C ratio of the early embryo, caused an exponential decline in DNA synthesis, which was suppressed by overexpression of RecQ4, Treslin,...

The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identifi ed Janus kinase 3 (JAK3) somatic-activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/ STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs. SIGNIFICANCE:Gene mutations causing NKTCL have not been fully identifi ed. Through exome sequencing, we identifi ed activating mutations of JAK3 that may play a signifi cant role in the pathogenesis of NKTCLs. Our fi ndings have important implications for the management of patients with NKTCLs.Cancer Discov; 2(7); 591-7.

Methylation of cytosine deoxynucleotides (dC5m) is a well-established epigenetic mark, but in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N-6-methyl-deoxyadenosine (dA6m) in vertebrate DNA, specifically in Xenopus laevis, but also in other species including mouse and human. Our methylome analysis reveals that dA6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.

BackgroundSnakebite is a global health issue and treatment with antivenom continues to be problematic. Brown snakes (genus Pseudonaja) are the most medically important group of Australian snakes and there is controversy over the dose of brown snake antivenom. We aimed to investigate the clinical and laboratory features of definite brown snake (Pseudonaja spp.) envenoming, and determine the dose of antivenom required.Methods and FindingThis was a prospective observational study of definite brown snake envenoming from the Australian Snakebite Project (ASP) based on snake identification or specific enzyme immunoassay for Pseudonaja venom. From January 2004 to January 2012 there were 149 definite brown snake bites [median age 42y (2–81y); 100 males]. Systemic envenoming occurred in 136 (88%) cases. All envenomed patients developed venom induced consumption coagulopathy (VICC), with complete VICC in 109 (80%) and partial VICC in 27 (20%). Systemic symptoms occurred in 61 (45%) and mild neurotoxicity in 2 (1%). Myotoxicity did not occur. Severe envenoming occurred in 51 patients (38%) and was characterised by collapse or hypotension (37), thrombotic microangiopathy (15), major haemorrhage (5), cardiac arrest (7) and death (6). The median peak venom concentration in 118 envenomed patients was 1.6 ng/mL (Range: 0.15–210 ng/mL). The median initial antivenom dose was 2 vials (Range: 1–40) in 128 patients receiving antivenom. There was no difference in INR recovery or clinical outcome between patients receiving one or more than one vial of antivenom. Free venom was not detected in 112/115 patients post-antivenom with only low concentrations (0.4 to 0.9 ng/ml) in three patients.ConclusionsEnvenoming by brown snakes causes VICC and over a third of patients had serious complications including major haemorrhage, collapse and microangiopathy. The results of this study support accumulating evidence that giving more than one vial of antivenom is unnecessary in brown snake envenoming.