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A. Palotie on työryhmän Int IBD Genetics Consortium jäsen. One goal of human genetics is to understand the genetic basis of disease, a challenge for diseases of complex inheritance because risk alleles are few relative to the vast set of benign variants. Risk variants are often sought by association studies in which allele frequencies in case subjects are contrasted with those from population-based samples used as control subjects. In an ideal world we would know population-level allele frequencies, releasing researchers to focus on case subjects. We argue this ideal is possible, at least theoretically, and we outline a path to achieving it in reality. If such a resource were to exist, it would yield ample savings and would facilitate the effective use of data repositories by removing administrative and technical barriers. We call this concept the Universal Control Repository Network (UNICORN), a means to perform association analyses without necessitating direct access to individual-level control data. Our approach to UNICORN uses existing genetic resources and various statistical tools to analyze these data, including hierarchical clustering with spectral analysis of ancestry; and empirical Bayesian analysis along with Gaussian spatial processes to estimate ancestry-specific allele frequencies. We demonstrate our approach using tens of thousands of control subjects from studies of Crohn disease, showing how it controls false positives, provides power similar to that achieved when all control data are directly accessible, and enhances power when control data are limiting or even imperfectly matched ancestrally. These results highlight how UNICORN can enable reliable, powerful, and convenient genetic association analyses without access to the individual-level data.

Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection., Highlights • We describe the genome sequence of two highly virulent ectromelia virus isolates. • The outbreak of ectromelia virus in USA was caused by Chinese viral isolates. • We describe a clade of ectromelia virus isolates from China. • We compare three different sequencing technologies to sequence large DNA viruses.

PCR probing of the genome of Campylobacter jejuni strain X using conserved capsular polysaccharide (CPS)-related genes allowed elucidation of a complete sequence of the respective gene cluster (cps). This is the largest known Campylobacter cps cluster (38 kb excluding flanking kps regions), which includes a number of genes not detected in other Campylobacter strains. Sequence analysis suggests genetic rearrangements both within and outside the cps gene cluster, a mechanism which may be responsible for mosaic organisation of sugar transferase-related genes leading to structural variability of the capsular polysaccharide (CPS).

Summary Background The emergence of meticillin-resistant Staphylococcus aureus (MRSA) that can persist in the community and replace existing hospital-adapted lineages of MRSA means that it is necessary to understand transmission dynamics in terms of hospitals and the community as one entity. We assessed the use of whole-genome sequencing to enhance detection of MRSA transmission between these settings. Methods We studied a putative MRSA outbreak on a special care baby unit (SCBU) at a National Health Service Foundation Trust in Cambridge, UK. We used whole-genome sequencing to validate and expand findings from an infection-control team who assessed the outbreak through conventional analysis of epidemiological data and antibiogram profiles. We sequenced isolates from all colonised patients in the SCBU, and sequenced MRSA isolates from patients in the hospital or community with the same antibiotic susceptibility profile as the outbreak strain. Findings The hospital infection-control team identified 12 infants colonised with MRSA in a 6 month period in 2011, who were suspected of being linked, but a persistent outbreak could not be confirmed with conventional methods. With whole-genome sequencing, we identified 26 related cases of MRSA carriage, and showed transmission occurred within the SCBU, between mothers on a postnatal ward, and in the community. The outbreak MRSA type was a new sequence type (ST) 2371, which is closely related to ST22, but contains genes encoding Panton-Valentine leucocidin. Whole-genome sequencing data were used to propose and confirm that MRSA carriage by a staff member had allowed the outbreak to persist during periods without known infection on the SCBU and after a deep clean. Interpretation Whole-genome sequencing holds great promise for rapid, accurate, and comprehensive identification of bacterial transmission pathways in hospital and community settings, with concomitant reductions in infections, morbidity, and costs. Funding UK Clinical Research Collaboration Translational Infection Research Initiative, Wellcome Trust, Health Protection Agency, and the National Institute for Health Research Cambridge Biomedical Research Centre.

Purpose Transcatheter aortic valve replacement (TAVR) has been successfully used to treat patients with failing aortic bioprostheses. Computed tomography (CT) is the usual method of pre-procedural imaging for TAVR in the native position; however, the optimal modality for valve-in-valve procedures has not been established. CT can assess intracardiac anatomy and is superior to cardiovascular magnetic resonance (CMR) in the assessment of coronary artery disease. However, CMR can provide superior haemodynamic information, does not carry the risk of ionising radiation, and may be performed without contrast in patients with renal insufficiency. In this study, we compared CT and CMR for the evaluation of TAVR in a small cohort of patients with existing aortic bioprostheses. Materials and methods 21 patients with aortic bioprostheses were prospectively evaluated by CT and CMR, as pre-assessment for TAVR; agreement between measurements of aortic geometries was assessed. Results 16/21 patients had aortic bioprostheses constructed with a metal ring, and 5/21 patients had a metal strut construction. Patients with metal struts had significant metal-artefact on CMR, which compromised image quality in this region. There was good agreement between CT and CMR measurements of aortic geometry. The mean difference ( d ) in annulus area-derived diameter was 0.5 mm (95% limits of agreement [L.A] 4.2 mm). There was good agreement between modalities for the cross-sectional area of the sinuses of valsalva ( d 0.5 cm 2 , L.A 1.4 cm 2 ), sinotubular junction ( d 0.9 cm 2 , L.A 1.5 cm 2 ), and ascending aorta ( d 0.6 cm 2 , L.A 1.4 cm 2 ). In patients without metal struts, the left coronary artery height d was 0.7 mm and L.A 2.8 mm. Conclusions Our analysis shows that CMR and CT measurements of aortic geometry show good agreement, including measurement of annulus size and coronary artery location, and thus provide the necessary anatomical information for valve-in-valve TAVR planning. However, in patients with metal strut aortic valve constructions, CT should be performed due to the presence of limiting metal artefacts on CMR. CMR may be considered as an appropriate alternative to CT in patients in whom iodinated contrast agents are contraindicated or where additional haemodynamic assessment with phase-contrast CMR is required.

Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.

The lung schistosomulum of Schistosoma mansoni is a validated target of protective immunity elicited in vaccinated mice. To identify genes expressed at this stage we constructed a microarray, representing 3088 contigs and singlets, with cDNA derived from in vitro cultured larvae and used it to screen RNA from seven life-cycle stages. Clustering of genes by expression profile across the life cycle revealed a number of membrane, membrane-associated and secreted proteins up-regulated at the lung stage, that may represent potential immune targets. Two promising secreted molecules have homology to antigens with vaccine and/or immunomodulatory potential in other helminths.

Comparative nematode genomics has thus far been largely constrained to the genus Caenorhabditis , but a huge diversity of other nematode species, and genomes, exist. The Brugia malayi genome is approximately 100 Mb in size, and distributed across five chromosome pairs. Previous genomic investigations have included definition of major repeat classes and sequencing of selected genes. We have generated over 18,000 sequences from the ends of large-insert clones from bacterial artificial chromosome libraries. These end sequences, totalling over 10 Mb of sequence, contain just under 8 Mb of unique sequence. We identified the known Mbo I and Hha I repeat families in the sequence data, and also identified several new repeats based on their abundance. Genomic copies of 17% of B. malayi genes defined by expressed sequence tags have been identified. Nearly one quarter of end sequences can encode peptides with significant similarity to protein sequences in the public databases, and we estimate that we have identified more than 2700 new B. malayi genes. Importantly, 459 end sequences had homologues in other organisms, but lacked a match in the completely sequenced genomes of Caenorhabditis briggsae and Caenorhabditis elegans , emphasising the role of gene loss in genome evolution. B. malayi is estimated to have over 18,500 protein-coding genes.

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.

Summary Background Whipple's disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. The causative agent, Tropheryma whipplei , is a Gram-positive bacterium about which little is known. Our aim was to investigate the biology of this organism by generating and analysing the complete DNA sequence of its genome. Methods We isolated and propagated T whipplei strain TW08/27 from the cerebrospinal fluid of a patient diagnosed with Whipple's disease. We generated the complete sequence of the genome by the whole genome shotgun method, and analysed it with a combination of automatic and manual bioinformatic techniques. Findings Sequencing revealed a condensed 925 938 bp genome with a lack of key biosynthetic pathways and a reduced capacity for energy metabolism. A family of large surface proteins was identified, some associated with large amounts of non-coding repetitive DNA, and an unexpected degree of sequence variation. Interpretation The genome reduction and lack of metabolic capabilities point to a host-restricted lifestyle for the organism. The sequence variation indicates both known and novel mechanisms for the elaboration and variation of surface structures, and suggests that immune evasion and host interaction play an important part in the lifestyle of this persistent bacterial pathogen.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Iron storage in the ferritin of Escherichia coli (EcFtnA) involves (1) the catalytic oxidation of 2Fe(II) at a dinuclear centre (sites A and B) and of a single Fe(II) at a nearby site C; (2) the formation of oxo-bridged Fe(III) dimers (designated ‘b’) at A and B and isolated Fe 3+ ions (‘c’) at site C; (3) the conversion of the oxo-bridged dimers to a second dimer form (‘e’) which is probably hydroxo-bridged; (4) the movement of iron into the iron storage cavity where Fe(III) ferrhydrite clusters (‘a’ and ‘d’) form, and (5) the provision on the surfaces of the mineral clusters of alternative sites for further Fe(II) oxidation as well as for the addition of Fe(III) from the dinuclear centres. Each of the iron species gives rise to a characteristic Mossbauer subspectrum and Mossbauer spectroscopy has been used to study the early stages of iron storage with the aid of competing metal ions Zn 2+ and Tb 3+ . The effects of adding these metal ions both before and after Fe(II) have been examined with both wild type EcFtnA and its site directed variants. These studies have provided evidence that both types of Fe(III) dimers are located at the dinuclear centre and that with time dimers ‘b’ are replaced by dimers ‘e’. The effects of the competing metals are different: Zn(II) has a lower affinity than Fe(III) for site C and does not displace it from C but is able to compete with both Fe(III) and Fe(II) for at least one of the dimer sites. Tb(III) competes with Fe(III) for site C displacing Fe(III) to the cavity to form clusters. The presence of these clusters may accelerate loss of Fe(III) from the dimer sites to the cavity.

The b acterioferritin-associated f erre d oxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene ( bfr ) encoding the iron-storage haemoprotein, bacterioferritin. The Bfd sequence resembles those of the ∼60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase. These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a [2Fe-2S] cluster. The Bfd protein was over-produced, purified, and characterised. Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides. Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a [2Fe-2S] 2+,+ centre (E 1/2 = −254 mV) having remarkably similar properties to the Fe-S cluster of NifU. Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression.

Minimum inhibitory concentrations (MIC) for Botrytis cinerea and Saccharomyces cerevisiae with ketoconazole were 10 μg ml− 1 and 20 μg ml− 1, respectively. The time-course of inhibition with treatment at the MIC was examined for each organism and the sterol profile determined. Botrytis cinerea initially accumulated 24-methylene-24(25)-dihydrolanosterol and then obtusifolione during growth arrest. Saccharomyces cerevisiae initially accumulated lanosterol with subsequent production of 3,6 diol, just prior to growth arrest.

Congenital stenosis of the pulmonary artery (PA) branches is an anomaly characterised by narrowed segments of one or more of the main or peripheral branches, and is commonly treated with balloon angioplasty or stenting. In patients with unilateral PA stenosis, the expected relationship between stenosis and reduced flow does not always appear to hold true. In our clinical experience, we have observed in some cases an equal flow between right and left PAs (RPA and LPA) despite the stenosis, or even higher flow in favour of the stenotic side. This may be explained by a mismatch in the distal pulmonary vascular resistance (PVR) between the two lungs [Cohn, 1976]. Patient-specific data and computational modelling were used to estimate PVR and understand the fluid dynamics in these patients before and after stent implantation.

Background & Aims The mechanisms by which specific mutations in NOD2 / CARD15 increase the risk for Crohn's disease (CD) are unclear. We identified proteins that interact with NOD2 and investigated them by expression, genetic, and functional analyses. Methods By using a yeast 2-hybrid screen of an intestinal epithelial library, we identified proteins that interact with NOD2 and confirmed the interactions in mammalian cells using co-immunoprecipitation. We used microarray analysis to analyze gene expression patterns in 302 intestinal biopsy samples (129 from patients with ulcerative colitis [UC], 106 with CD, and 67 controls). Eighty single-nucleotide polymorphisms within the genes that encoded 6 interacting proteins were genotyped in a discovery cohort (869 cases of inflammatory bowel disease [IBD], 885 controls) and a replication cohort (504 patients with IBD, 713 controls). We investigated interaction between transducin-like enhancer of split 1 (TLE1) and NOD2 in HEK293 cells. Results We identified 6 NOD2-interacting proteins (TLE1, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 [GALNT2], HIV-1 Tat interactive protein [HTATIP], Vimentin, fission 1 (mitochondrial outer membrane) homolog [FIS1], and protein phosphatase 2, regulatory subunit B′, epsilon isoform [PPP2R5E]). Of these, expression of GALNT2 (CD, P = .004) and vimentin (CD, P = .006; UC, P = .0025) was altered in patients with IBD compared with controls. Single-nucleotide polymorphisms within TLE1 were associated with susceptibility to CD, specifically with ileal disease (rs6559629, P = 3.1 × 10 −5 ; odds ratio, 1.45). The TLE1 risk allele is required for susceptibility to CD in carriers of NOD2 mutations. In cells, TLE1 and NOD2 co-localized around the nuclear membrane and TLE1 inhibited activation of nuclear factor-κB by NOD2. Conclusions Epistatic and biological interactions between TLE1 and NOD2 are involved in IBD pathogenesis. NOD2 might be involved in a series of pathways such as epigenetic regulation of expression (via TLE1 and HTATIP), biosynthesis of mucin (via GALNT2), apoptosis (via PPP2R5E and FIS1), and integrity of the intracellular cytoskeleton (vimentin).