The complex portal--an encyclopaedia of macromolecular complexes.

The IntAct molecular interaction database has created a new, free, open-source, manually curated resource, the Complex Portal (www.ebi.ac.uk/intact/complex), through which protein complexes from major model organisms are being collated and made available for search, viewing and download. It has been built in close collaboration with other bioinformatics services and populated with data from ChEMBL, MatrixDB, PDBe, Reactome and UniProtKB. Each entry contains information about the participating molecules (including small molecules and nucleic acids), their stoichiometry, topology and structural assembly. Complexes are annotated with details about their function, properties and complex-specific Gene Ontology (GO) terms. Consistent nomenclature is used throughout the resource with systematic names, recommended names and a list of synonyms all provided. The use of the Evidence Code Ontology allows us to indicate for which entries direct experimental evidence is available or if the complex has been inferred based on homology or orthology. The data are searchable using standard identifiers, such as UniProt, ChEBI and GO IDs, protein, gene and complex names or synonyms. This reference resource will be maintained and grow to encompass an increasing number of organisms. Input from groups and individuals with specific areas of expertise is welcome.

The MIntAct project--IntAct as a common curation platform for 11 molecular interaction databases.

IntAct (freely available at http://www.ebi.ac.uk/intact) is an open-source, open data molecular interaction database populated by data either curated from the literature or from direct data depositions. IntAct has developed a sophisticated web-based curation tool, capable of supporting both IMEx- and MIMIx-level curation. This tool is now utilized by multiple additional curation teams, all of whom annotate data directly into the IntAct database. Members of the IntAct team supply appropriate levels of training, perform quality control on entries and take responsibility for long-term data maintenance. Recently, the MINT and IntAct databases decided to merge their separate efforts to make optimal use of limited developer resources and maximize the curation output. All data manually curated by the MINT curators have been moved into the IntAct database at EMBL-EBI and are merged with the existing IntAct dataset. Both IntAct and MINT are active contributors to the IMEx consortium (http://www.imexconsortium.org).

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells.

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1ß, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein "knock-down" and allows temporal control of stable signaling components at the protein level.

Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells.

Notch signalling occurs via direct cell-cell interactions and plays an important role in linking the fates of neighbouring cells. There are four different mammalian Notch receptors that can be activated by five cell surface ligands. The ability to inhibit specific Notch receptors would help identify the roles of individual family members and potentially provide a means to study and control cell differentiation. Anti-Notch antibodies in the form of single chain Fvs were generated from an antibody phage display library by selection on either the ligand binding domain or the negative regulatory region (NRR) of Notch1 and Notch2. Six antibodies targeting the NRR of Notch1 and four antibodies recognising the NRR of Notch2 were found to prevent receptor activation in cell-based luciferase reporter assays. These antibodies were potent, highly specific inhibitors of individual Notch receptors and interfered with endogenous signalling in stem cell systems of both human and mouse origin. Antibody-mediated inhibition of Notch efficiently down-regulated transcription of the immediate Notch target gene hairy and enhancer of split 5 (Hes5) in both mouse and human neural stem cells and revealed a redundant regulation of Hes5 in these cells as complete down-regulation was seen only after simultaneous blocking of Notch1 and Notch2. In addition, these antibodies promoted differentiation of neural stem cells towards a neuronal fate. In contrast to the widely used small molecule γ-secretase inhibitors, which block all 4 Notch receptors (and a multitude of other signalling pathways), antibodies allow blockade of individual Notch family members in a highly specific way. Specific inhibition will allow examination of the effect of individual Notch receptors in complex differentiation schemes regulated by the co-ordinated action of multiple signalling pathways.

VE-CADHERIN is expressed transiently in early ISL1+ cardiovascular progenitor cells and facilitates cardiac differentiation.

Adherens junctions (AJs) provide adhesive properties through cadherins and associated cytoplasmic catenins and participate in morphogenetic processes. We examined AJs formed between ISL1+ cardiovascular progenitor cells during differentiation of embryonic stem cells (ESCs) in vitro and in mouse embryogenesis in vivo. We found that, in addition to N-CADHERIN, a percentage of ISL1+ cells transiently formed vascular endothelial (VE)-CADHERIN-mediated AJs during in vitro differentiation on days 4 and 5, and the same pattern was observed in vivo. Fluorescence-activated cell sorting (FACS) analysis extended morphological data showing that VE-CADHERIN+/ISL1+ cells constitute a significant percentage of cardiac progenitors on days 4 and 5. The VE-CADHERIN+/ISL1+ cell population represented one-third of the emerging FLK1+/PDGFRa+ cardiac progenitor cells (CPCs) for a restricted time window (days 4-6). Ablation of VE-CADHERIN during ESC differentiation results in severe inhibition of cardiac differentiation. Disruption of all classic cadherins in the VE-CADHERIN+ population via a cadherin dominant-negative mutant's expression resulted in a dramatic decrease in the ISL1+ population and inhibition of cardiac differentiation.

Microinjection of targeted embryonic stem cells and establishment of knockout mouse lines for Fmo genes.

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.

Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

The phenotype of a knockout mouse identifies flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic ageing.

We report the production and metabolic phenotype of a mouse line in which the Fmo5 gene is disrupted. In comparison with wild-type (WT) mice, Fmo5(-/-) mice exhibit a lean phenotype, which is age-related, becoming apparent after 20 weeks of age. Despite greater food intake, Fmo5(-/-) mice weigh less, store less fat in white adipose tissue (WAT), have lower plasma glucose and cholesterol concentrations and enhanced whole-body energy expenditure, due mostly to increased resting energy expenditure, with no increase in physical activity. An increase in respiratory exchange ratio during the dark phase, the period in which the mice are active, indicates a switch from fat to carbohydrate oxidation. In comparison with WT mice, the rate of fatty acid oxidation in Fmo5(-/-) mice is higher in WAT, which would contribute to depletion of lipid stores in this tissue, and lower in skeletal muscle. Five proteins were down regulated in the liver of Fmo5(-/-) mice: aldolase B, ketohexokinase and cytosolic glycerol 3-phosphate dehydrogenase (GPD1) are involved in glucose or fructose metabolism and GPD1 also in production of glycerol 3-phosphate, a precursor of triglyceride biosynthesis; HMG-CoA synthase 1 is involved in cholesterol biosynthesis; and malic enzyme 1 catalyzes the oxidative decarboxylation of malate to pyruvate, in the process producing NADPH for use in lipid and cholesterol biosynthesis. Down regulation of these proteins provides a potential explanation for the reduced fat deposits and lower plasma cholesterol characteristic of Fmo5(-/-) mice. Our results indicate that disruption of the Fmo5 gene slows metabolic ageing via pleiotropic effects.

Microinjection of Targeted Embryonic Stem Cells and Establishment of Knockout Mouse Lines for Fmo Genes.

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.