Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic ß-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

The Xenopus phenotype ontology: bridging model organism phenotype data to human health and development.

BACKGROUND: Ontologies of precisely defined, controlled vocabularies are essential to curate the results of biological experiments such that the data are machine searchable, can be computationally analyzed, and are interoperable across the biomedical research continuum. There is also an increasing need for methods to interrelate phenotypic data easily and accurately from experiments in animal models with human development and disease. RESULTS: Here we present the Xenopus phenotype ontology (XPO) to annotate phenotypic data from experiments in Xenopus, one of the major vertebrate model organisms used to study gene function in development and disease. The XPO implements design patterns from the Unified Phenotype Ontology (uPheno), and the principles outlined by the Open Biological and Biomedical Ontologies (OBO Foundry) to maximize interoperability with other species and facilitate ongoing ontology management. Constructed in Web Ontology Language (OWL) the XPO combines the existing uPheno library of ontology design patterns with additional terms from the Xenopus Anatomy Ontology (XAO), the Phenotype and Trait Ontology (PATO) and the Gene Ontology (GO). The integration of these different ontologies into the XPO enables rich phenotypic curation, whilst the uPheno bridging axioms allows phenotypic data from Xenopus experiments to be related to phenotype data from other model organisms and human disease. Moreover, the simple post-composed uPheno design patterns facilitate ongoing XPO development as the generation of new terms and classes of terms can be substantially automated. CONCLUSIONS: The XPO serves as an example of current best practices to help overcome many of the inherent challenges in harmonizing phenotype data between different species. The XPO currently consists of approximately 22,000 terms and is being used to curate phenotypes by Xenbase, the Xenopus Model Organism Knowledgebase, forming a standardized corpus of genotype-phenotype data that can be directly related to other uPheno compliant resources.

Correction: Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice.

[This corrects the article DOI: 10.1371/journal.pgen.1002114.].

A trans-acting protein effect causes severe eye malformation in the Mp mouse.

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.

Gain-of-function mutations of ARHGAP31, a Cdc42/Rac1 GTPase regulator, cause syndromic cutis aplasia and limb anomalies.

Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development.

Loss of the BMP antagonist, SMOC-1, causes Ophthalmo-acromelic (Waardenburg Anophthalmia) syndrome in humans and mice.

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5-16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg(-/-)) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

Uncertainty in end-of-life care.

PURPOSE OF REVIEW: Uncertainty surrounding medical decision-making is particularly important during end-of-life decision-making. Doubts about the patient's best interests and prognostic accuracy may lead to conflict. RECENT FINDINGS: Many authors have suggested recently that medical attitudes to uncertainty need review. It is inappropriate to avoid discussion of uncertainty during end-of-life care and American literature suggests that patients and families accept uncertainty in end-of-life discussions. Recently, authors have advocated the concept of 'Practical Certainty' accepting that absolute certainty is rarely possible in end-of-life decision-making and openly acknowledging that the physicians are as certain as they can be in the circumstances. Allowing time to provide acceptance of a palliative care pathway and using the collective wisdom of colleagues improves the accuracy of prediction and reduces conflict at the end of life. SUMMARY: The implications of this review are that doctors should not avoid discussing uncertainty in end-of-life conversations and the article provides some recommendations for minimizing conflict arising from end-of-life discussion.

Xenbase: key features and resources of the Xenopus model organism knowledgebase.

Xenbase (https://www.xenbase.org/), the Xenopus model organism knowledgebase, is a web-accessible resource that integrates the diverse genomic and biological data from research on the laboratory frogs Xenopus laevis and Xenopus tropicalis. The goal of Xenbase is to accelerate discovery and empower Xenopus research, to enhance the impact of Xenopus research data, and to facilitate the dissemination of these data. Xenbase also enhances the value of Xenopus data through high-quality curation, data integration, providing bioinformatics tools optimized for Xenopus experiments, and linking Xenopus data to human data, and other model organisms. Xenbase also plays an indispensable role in making Xenopus data interoperable and accessible to the broader biomedical community in accordance with FAIR principles. Xenbase provides annotated data updates to organizations such as NCBI, UniProtKB, Ensembl, the Gene Ontology consortium, and most recently, the Alliance of Genomic Resources, a common clearing house for data from humans and model organisms. This article provides a brief overview of key and recently added features of Xenbase. New features include processing of Xenopus high-throughput sequencing data from the NCBI Gene Expression Omnibus; curation of anatomical, physiological, and expression phenotypes with the newly created Xenopus Phenotype Ontology; Xenopus Gene Ontology annotations; new anatomical drawings of the Normal Table of Xenopus development; and integration of the latest Xenopus laevis v10.1 genome annotations. Finally, we highlight areas for future development at Xenbase as we continue to support the Xenopus research community.

EMAGE mouse embryo spatial gene expression database: 2010 update.

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (approximately 19,000 gene) 'EURExpress' dataset into EMAGE.

3D expression patterns of cell cycle genes in the developing chick wing and comparison with expression patterns of genes implicated in digit specification.

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N-myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh.

Disruption of ST5 is associated with mental retardation and multiple congenital anomalies.

BACKGROUND: The authors observed a patient with a cryptic subtelomeric de novo balanced translocation 46,XY.ish t(11;20)(p15.4;q13.2) presenting with severe mental retardation, muscular hypotonia, seizures, bilateral sensorineural hearing loss, submucous cleft palate, persistent ductus Botalli, unilateral cystic kidney dysplasia and frequent infections. METHODS AND RESULTS: Fluorescence in situ hybridisation mapping and sequencing of the translocation breakpoints showed that no known genes are disrupted at 20q13.2 and that ST5 (suppression of tumorigenicity 5; MIM 140750) is disrupted on 11p15.4. By quantitative PCR from different human tissues, the authors found ST5 to be relatively evenly expressed in fetal tissues. ST5 expression was more pronounced in adult brain, kidney and muscle than in the corresponding fetal tissues, whereas expression in other tissues was generally lower than in the fetal tissue. Using RNA in situ hybridisation in mouse, the authors found that St5 is expressed in the frontal cortex during embryonic development. In adult mouse brain, expression of St5 was especially high in the hippocampal area and cerebellum. CONCLUSION: Hence, the authors suppose that ST5 plays an important role in central nervous system development probably due to disturbance of DENN-domain-mediated vesicle formation and neurotransmitter trafficking. Thus, these findings implicate ST5 in the aetiology of mental retardation, seizures and multiple congenital anomalies.

Integrating technologies for comparing 3D gene expression domains in the developing chick limb.

Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using whole amount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

Xenbase: Facilitating the Use of Xenopus to Model Human Disease.

At a fundamental level most genes, signaling pathways, biological functions and organ systems are highly conserved between man and all vertebrate species. Leveraging this conservation, researchers are increasingly using the experimental advantages of the amphibian Xenopus to model human disease. The online Xenopus resource, Xenbase, enables human disease modeling by curating the Xenopus literature published in PubMed and integrating these Xenopus data with orthologous human genes, anatomy, and more recently with links to the Online Mendelian Inheritance in Man resource (OMIM) and the Human Disease Ontology (DO). Here we review how Xenbase supports disease modeling and report on a meta-analysis of the published Xenopus research providing an overview of the different types of diseases being modeled in Xenopus and the variety of experimental approaches being used. Text mining of over 50,000 Xenopus research articles imported into Xenbase from PubMed identified approximately 1,000 putative disease- modeling articles. These articles were manually assessed and annotated with disease ontologies, which were then used to classify papers based on disease type. We found that Xenopus is being used to study a diverse array of disease with three main experimental approaches: cell-free egg extracts to study fundamental aspects of cellular and molecular biology, oocytes to study ion transport and channel physiology and embryo experiments focused on congenital diseases. We integrated these data into Xenbase Disease Pages to allow easy navigation to disease information on external databases. Results of this analysis will equip Xenopus researchers with a suite of experimental approaches available to model or dissect a pathological process. Ideally clinicians and basic researchers will use this information to foster collaborations necessary to interrogate the development and treatment of human diseases.

Navigating Xenbase: An Integrated Xenopus Genomics and Gene Expression Database.

Xenbase is the Xenopus model organism database ( www.xenbase.org ), a web-accessible resource that integrates the diverse genomic and biological data for Xenopus research. It hosts a variety of content including current and archived genomes for both X. laevis and X. tropicalis, bioinformatic tools for comparative genetic analyses including BLAST and GBrowse, annotated Xenopus literature, and catalogs of reagents including antibodies, ORFeome clones, morpholinos, and transgenic lines. Xenbase compiles gene-specific pages which include manually curated gene expression images, functional information including gene ontology (GO), disease associations, and links to other major data sources such as NCBI:Entrez, UniProtKB, and Ensembl. We also maintain the Xenopus Anatomy Ontology (XAO) which describes anatomy throughout embryonic development. This chapter provides a full description of the many features of Xenbase, and offers a guide on how to use various tools to perform a variety of common tasks such as identifying nucleic acid or protein sequences, finding gene expression patterns for specific genes, stages or tissues, identifying literature on a specific gene or tissue, locating useful reagents and downloading our extensive content, including Xenopus gene-Human gene disease mapping files.

Intensive care in Australia and New Zealand.

This article provides an up-to-date account of intensive care in Australia and New Zealand. Overall, intensive care medicine in Australia and New Zealand is in a healthy state. It began early and has developed into a distinct and strong medical specialty. There are well-developed organizations dealing with all aspects of the specialty. The ICU is recognized as an expensive and limited resource. Overall, intensivists in these two countries have good control of it and have learned to use it wisely.

The prevention of anaphylactoid reactions to iodinated radiological contrast media: a systematic review.

BACKGROUND: Anaphylactoid reactions to iodinated contrast media are relatively common and potentially life threatening. Opinion is divided as to the utility of medications for preventing these reactions. We performed a systematic review to assess regimes for the prevention of anaphylactoid reactions to iodinated contrast media. METHODS: Searches for studies were conducted in the Medline, EMBASE, CINAHL and CENTRAL databases. Bibliographies of included studies and review articles were examined and experts were contacted. Randomised clinical trials that examined agents given prior to iodinated contrast material for the prevention of anaphylactoid reactions were included in the review. The validity of the included studies was examined using a component approach. RESULTS: Six studies met the inclusion criteria, but only one of these fulfilled all of the validity criteria. There were four studies that examined the use of H1 antihistamines, each was used to prevent anaphylactoid reactions to ionic contrast. The random effects pooled relative risk demonstrated a significant reduction in the overall rate of anaphylactoid reactions (RR = 0.4, 95% CI 0.18-0.9, p = 0.027). There were insufficient studies to produce a pooled statistic for the use of corticosteroids, however regimes of steroids (methylprednisolone 32 mg) given at least six hours and again two hours prior to the administration of contrast suggested a reduction in the incidence of anaphylactoid reactions. CONCLUSION: In conclusion, there are few high quality randomised clinical trials that have addressed the question of the optimal methods to prevent allergic type reactions to iodinated radiological contrast media. Allowing for these limitations, the results suggest that H1 antihistamines given immediately prior to the administration of ionic contrast may be useful in preventing reactions to ionic contrast and are suggestive of a protective effect of corticosteroids when given in two doses at least six hours prior and again two hours prior to the administration of contrast, both ionic and non-ionic. These agents should be considered for use in patients who are at high risk of an anaphylactoid reaction to contrast media and for who prophylactic therapy is considered necessary. Further research is needed before definitive recommendations can be made.

Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

Anaphylaxis to anaesthetic drugs.

Severe anaphylactic reactions during anaesthesia increased dramatically in incidence in the mid 1970s. Studies performed in our unit over the subsequent 25 years demonstrated the involvement of IgE in these reactions and the value and safety of intradermal and prick testing in the diagnosis and determination of the drug responsible. Radioimmunoassay studies demonstrated that neuromuscular blocking drugs produce anaphylaxis by cross-linking IgE molecules via their substituted ammonium groups. The IgE binding of these drugs leads to a high incidence of cross sensitivity. Mast cell tryptase measurement is highly sensitive and specific for anaphylaxis although it can be elevated in reactions due to direct histamine release. The reactions are unpredictable from the history. The heart is rarely a target organ in human anaphylaxis although the diseased heart is more likely to fail or produce serious arrhythmias than the normal heart. Colloid solutions produce a better response than crystalloid solutions in the treatment of hypotension. Anaesthetic allergy persists up to 27 years. Subsequent anaesthesia based on the findings of skin testing is usually safe.

Prevention of anaphylactic reactions to anaesthetic drugs.

Although screening tests to prevent anaphylaxis during anaesthesia have been advocated, such tests are unlikely to have significant impact on reducing the incidence of anaphylaxis during anaesthesia. This is due to the low prevalence of the disease, the diversity of drugs used in anaesthesia and the incidence of false positive and negative tests. The suggested risk factors of allergy, i.e. atopy, asthma, family history, female sex, previous exposure, vasectomy, use of zinc protamine sulfate insulin and allergy to cosmetics, eggs, fish and non-anaesthetic drugs are not valid. Although all have theoretical or real associations with anaphylaxis during anaesthesia the majority of patients with such a history undergo uneventful anaesthesia. Fruit allergy, anaphylaxis to cephalosporins and penicillin, barbiturate allergy, gelatin allergy and allergy to metabisulphite and eggs require consideration in avoiding particular drugs. The incidence of anaesthetic anaphylaxis can be reduced by avoiding latex exposure in patients with spina bifida or latex allergy, and preventing second reactions in patients with a history of anaphylaxis, or major undiagnosed or undocumented adverse events during anaesthesia. Determining the cause of an adverse event and the drug responsible, and adequately communicating those findings can reduce second reactions. Avoiding neuromuscular blocking drugs (NMBDs) in patients who have reacted to an NMBD, and use of non-intravenous techniques should also reduce the incidence of second reactions. Desensitisation, and blocking with monovalent quaternary ammonium compounds may allow improved safety of NMBDs and pretreatment with antihistamines and corticosteroids may block or ameliorate the severity of reactions, but there is currently little evidence to support their routine use.