Grounding annotations in published literature with an emphasis on the functional roles used in metabolic models

Accurate genome annotations in databases are a critical resource available to the scientific community for analysis and research. Inaccurate and inconsistent annotations exist as a result of errors generated from mass automated annotation, and currently act as a barrier to the application of bioinformatics. The purpose of this effort was to improve the SEED by improving the connection of functional roles to literature references. Direct literature references (DLits), found through searches of PubMed and other online databases such as SwissProt, were attached to protein sequences within the PubSEED to provide literature support for the roughly 2,500 distinct functional roles used to construct metabolic models within the Model SEED. Only DLits in which a researcher asserted the function of a protein were attached to sequences. Starting from a list of 1,072 functional roles that did not previously have DLit support, we were able to connect sequences to literature for 655 functional roles, at least 484 of which were in the original list of unsupported roles. When added to the existing set of sequences having DLits, the resulting set of DLit-sequence pairs (the foundation set) now connects approximately 4,300 DLits to approximately 5,600 distinct protein sequences obtained from approximately 16,000 genes (some of these genes have identical protein sequences). From the foundation set, we construct projection sets such that each set contains one member of the foundation set and projections of its functional role onto similar genes. The projection sets revealed 120 inconsistent annotations within the SEED. Two types of inconsistencies were corrected through manual annotation in the PubSEED: instances in which two identical protein sequences had been annotated with different functions, and instances when projected functions contradicted previous annotations. 26,785 changes to gene function assignment, 219 of which were to previously uncharacterized proteins, resulted in a more consistent and accurate set of input data from which to construct revised metabolic models within the Model SEED. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13205-011-0039-z) contains supplementary material, which is available to authorized users

Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect® Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species' repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B-cells are able to go through complete development

Interferon-induced Transmembrane Protein 1 restricts replication of virus that enter cells via the plasma membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections

Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions

201TI Myocardial SPECT and ß-Endorphin Levels in Patients with Suspected Silent Ischemia

Today silent myocardial ischemia (SMI) is a well-recognized phenomenon. However, in the absence of clinical signs suggesting coronary artery disease (CAD), a streamlined diagnostic approach for precise clarification has proved to be difficult. Sensitivity and specificity of ergometric results are rather poor in symptom-free patients. Thus the question arises, whether the necessity of coronary angiography can be established more precisely by 201TI myocardial SPECT in these patients. Treadmill exercise according to the Bruce protocol, 201TI myocardial SPECT and coronary angiography were performed in a total of 106 patients with suspected SMI. In group I (high probability of ischemia; n = 46), reversible defects detected by SPECT correlated well with significant stenoses and irreversible defects with subtotal stenoses or complete occlusions. SPECT sensitivity in the detection of ischemia was 91 %, its specificity 96%. In group II (low probability of ischemia; n = 60), SPECT sensitivity was as high as in group I (94%) but due to a high number of false-positive results (e. g. cardiomyopathy) specificity was only 75%. However, SPECT was superior to exercise ECG (sensitivity 70%; specificity 56%) in the detection of SMI. In addition, ß-endorphin levels were determined in 180 healthy subjects, 37 patients with symptomatic CAD and in 34 patients with SMI before and during maximum exercise. Exercise values in patients with SMI were significantly higher than in healthy subjects or in patients with symptomatic CAD.</jats:p