A web-based pilot study of inter-pathologist reproducibility using the ISHLT 2004 working formulation for biopsy diagnosis of cardiac allograft rejection: The European experience.

BACKGROUND: The aim of this study was to assess, at the European level and using digital technology, the inter-pathologist reproducibility of the ISHLT 2004 system and to compare it with the 1990 system We also assessed the reproducibility of the morphologic criteria for diagnosis of antibody-mediated rejection detailed in the 2004 grading system. METHODS: The hematoxylin-eosin-stained sections of 20 sets of endomyocardial biopsies were pre-selected and graded by two pathologists (A.A. and M.B.) and digitized using a telepathology digital pathology system (Aperio ImageScope System; for details refer to http://aperio.com/). Their diagnoses were considered the index diagnoses, which covered all grades of acute cellular rejection (ACR), early ischemic lesions, Quilty lesions, late ischemic lesions and (in the 2005 system) antibody-mediated rejection (AMR). Eighteen pathologists from 16 heart transplant centers in 7 European countries participated in the study. Inter-observer reproducibility was assessed using Fleiss's kappa and Krippendorff's alpha statistics. RESULTS: The combined kappa value of all grades diagnosed by all 18 pathologists was 0.31 for the 1990 grading system and 0.39 for the 2005 grading system, with alpha statistics at 0.57 and 0.55, respectively. Kappa values by grade for 1990/2005, respectively, were: 0 = 0.52/0.51; 1A/1R = 0.24/0.36; 1B = 0.15; 2 = 0.13; 3A/2R = 0.29/0.29; 3B/3R = 0.13/0.23; and 4 = 0.18. For the 2 cases of AMR, 6 of 18 pathologists correctly suspected AMR on the hematoxylin-eosin slides, whereas, in each of 17 of the 18 AMR-negative cases a small percentage of pathologists (range 5% to 33%) overinterpreted the findings as suggestive for AMR. CONCLUSIONS: Reproducibility studies of cardiac biopsies by pathologists in different centers at the international level were feasible using digitized slides rather than conventional histology glass slides. There was a small improvement in interobserver agreement between pathologists of different European centers when moving from the 1990 ISHLT classification to the "new" 2005 ISHLT classification. Morphologic suspicion of AMR in the 2004 system on hematoxylin-eosin-stained slides only was poor, highlighting the need for better standardization of morphologic criteria for AMR. Ongoing educational programs are needed to ensure standardization of diagnosis of both acute cellular and antibody-mediated rejection.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

MR spectroscopy of the human brain with enhanced signal intensity at ultrashort echo times on a clinical platform at 3T and 7T.

Recently, the spin-echo full-intensity acquired localized (SPECIAL) spectroscopy technique was proposed to unite the advantages of short TEs on the order of milliseconds (ms) with full sensitivity and applied to in vivo rat brain. In the present study, SPECIAL was adapted and optimized for use on a clinical platform at 3T and 7T by combining interleaved water suppression (WS) and outer volume saturation (OVS), optimized sequence timing, and improved shimming using FASTMAP. High-quality single voxel spectra of human brain were acquired at TEs below or equal to 6 ms on a clinical 3T and 7T system for six volunteers. Narrow linewidths (6.6 +/- 0.6 Hz at 3T and 12.1 +/- 1.0 Hz at 7T for water) and the high signal-to-noise ratio (SNR) of the artifact-free spectra enabled the quantification of a neurochemical profile consisting of 18 metabolites with Cramér-Rao lower bounds (CRLBs) below 20% at both field strengths. The enhanced sensitivity and increased spectral resolution at 7T compared to 3T allowed a two-fold reduction in scan time, an increased precision of quantification for 12 metabolites, and the additional quantification of lactate with CRLB below 20%. Improved sensitivity at 7T was also demonstrated by a 1.7-fold increase in average SNR (= peak height/root mean square [RMS]-of-noise) per unit-time.

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

MyHits: a new interactive resource for protein annotation and domain identification.

The MyHits web server (http://myhits.isb-sib.ch) is a new integrated service dedicated to the annotation of protein sequences and to the analysis of their domains and signatures. Guest users can use the system anonymously, with full access to (i) standard bioinformatics programs (e.g. PSI-BLAST, ClustalW, T-Coffee, Jalview); (ii) a large number of protein sequence databases, including standard (Swiss-Prot, TrEMBL) and locally developed databases (splice variants); (iii) databases of protein motifs (Prosite, Interpro); (iv) a precomputed list of matches ('hits') between the sequence and motif databases. All databases are updated on a weekly basis and the hit list is kept up to date incrementally. The MyHits server also includes a new collection of tools to generate graphical representations of pairwise and multiple sequence alignments including their annotated features. Free registration enables users to upload their own sequences and motifs to private databases. These are then made available through the same web interface and the same set of analytical tools. Registered users can manage their own sequences and annotations using only web tools and freeze their data in their private database for publication purposes.

Recrystallisation rims in zircon (Valle d'Arbedo, Switzerland): an integrated cathodoluminescence, LA-ICP-MS, SHRIMP, and TEM study

Recrystallization rims are a common feature of zircon crystals that underwent metamorphism. We present a microstructural and microchemical study of partially recrystallized zircon grains collected in polymetamorphic migmatites (Valle d'Arbedo, Ticino, Switzerland). The rims are bright in cathodo-luminescence (CL), with sharp and convex contacts characterized by inward-penetrating embayments transgressing igneous zircon cores. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) data and transmission electron microscopy (TEM) imaging indicate that the rims are chemically and microstructurally different from the cores. The rims are strongly depleted in REE, with concentrations up to two orders of magnitude lower than in the cores, indicating a significant loss of REE during zircon recrystallization. Enrichment in non-formula elements, such as Ca, has not been observed in the rims. The microstructure of zircon cores shows a dappled intensity at and below the 100 nm scale, possibly due to radiation damage. Other defects such as pores and dislocations are absent in the core except at healed cracks. Zircon rims are mostly dapple-free, but contain nanoscale pores and strain centers, interpreted as fluid inclusions and chemical residues, respectively. Sensitive high-resolution ion microprobe (SHRIMP) U-Pb ages show that the recrystallization of the rims took place >200 Ma ago when the parent igneous zircon was not metamict. The chemical composition and the low-Ti content of the rims indicate that they form at sub-solidus temperatures (550-650 degrees C). Recrystallization rims in Valle d'Arbedo zircon are interpreted as the result of the migration of chemical reaction fronts in which fluid triggered in situ and contemporaneous interface-coupled dissolution-reprecipitation mechanisms. This study indicates that strong lattice strain resulting from the incorporation of a large amount of impurities and structural defects is not a necessary condition for zircon to recrystallize. Our observations suggest that the early formation of recrystallization rims played a major role in preserving zircon from the more recent Alpine metamorphic overprint.

SwissDock, a protein-small molecule docking web service based on EADock DSS.

Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community.