On the Accuracy of Equivalent Antenna Representations

The accuracy of two equivalent antenna representations, near-field sources and far-field sources, are evaluated for an antenna installed on a simplified platform in a series of case studies using different configurations of equivalent antenna representations. The accuracy is evaluated in terms of installed far-fields and surface currents on the platform. The results show large variations between configurations. The root-mean-square installed far-field error is 4.4% for the most accurate equivalent representation. When using far-field sources, the design parameters have a large influence of the achieved accuracy. There is also a varying accuracy depending on the type of numerical method used. Based on the results, some recommendations on the choice of sub-domain for the equivalent antenna representation are given. In industrial antenna applications, the accuracy in determining e.g. installed far-fields and antenna isolation on large platforms are critical. Equivalent representations can reduce the fine-detail complexity of antennas and thus give an efficient numerical descriptions to be used in large-scale simulations. The results in this paper can be used as a guideline by antenna designers or system engineers when using equivalent sources

DIANA—algorithmic improvements for analysis of data-independent acquisition MS data

Motivation: Data independent acquisition mass spectrometry has emerged as a reproducible and sensitive alternative in quantitative proteomics, where parsing the highly complex tandem mass spectra requires dedicated algorithms. Recently, targeted data extraction was proposed as a novel analysis strategy for this type of data, but it is important to further develop these concepts to provide quality-controlled, interference-adjusted and sensitive peptide quantification. Results: We here present the algorithm DIANA and the classifier PyProphet, which are based on new probabilistic sub-scores to classify the chromatographic peaks in targeted data-independent acquisition data analysis. The algorithm is capable of providing accurate quantitative values and increased recall at a controlled false discovery rate, in a complex gold standard dataset. Importantly, we further demonstrate increased confidence gained by the use of two complementary data-independent acquisition targeted analysis algorithms, as well as increased numbers of quantified peptide precursors in complex biological samples. Availability and implementation: DIANA is implemented in scala and python and available as open source (Apache 2.0 license) or pre-compiled binaries from http://quantitativeproteomics.org/diana. PyProphet can be installed from PyPi (https://pypi.python.org/pypi/pyprophet). Supplementary information: Supplementary data are available at Bioinformatics onlin

IISEE Tutor Community : Ett funktionellt produktutvecklingsarbete av en systematisk helhet som betjänar och aktiverar företagets instruktörer

Detta examensarbete är av typen funktionellt produktutvecklingsarbete samt ett beställningsarbete av det finländska företaget IISEE. IISEE är både ett inomhuscyklingskoncept (spinning) samt utbildningsorganisation för inomhuscyklingsinstruktörer. Företaget har varit verksamt sedan 2008. I Finland har det sammanlagt utbildats omkring 1000 IISEE-instruktörer varav cirka 200 är aktiva idag. Syftet med arbetet är att utveckla en produkt (IISEE Tutor Community) som grundar sig på tutorverksamhet och fungerar som en sammanhängande systematisk helhet. Produktens främsta uppgift är att öka aktiviteten och växelverkan mellan IISEE-instruktörerna samt betjäna alla parter som berörs av produkten. Problemställningen i arbetet är följande: Vilka egenskaper anser IISEE-instruktörerna att är nödvändiga för produkten. Vad är det som IISEE-instruktörerna önskar och eftertraktar? Vad är möjligt samt realistiskt att förverkliga och vad är inte gällande produkten? Hur kan nuvarande system utnyttjas och utvecklas i och med lanseringen av produkten? Vad innebär en välfungerande tutorverksamhet? Hur väljs de IISEE-instruktörer som blir tutorer? Kan tutorerna belönas för sin insats i annan form än pengar? Examensarbetet består av en teoretisk del där inomhuscykling, IISEE som företag och tutorverksamhetens innebörd presenteras. Efter detta följer problemformuleringen och metod. Metoden som använts i detta arbete är processbeskrivning och grundar sig på Vilkka och Airaksinens bok Toiminnallinen opinnäytetyö (2003). Själva produktutvecklingsprocessen följer fem stycken faser i ett produktutvecklingsarbete och baserar sig på Jämsä och Manninens bok Osaamisen tuotteistaminen sosiaali- ja terveysalalla (2000). Som stöd för framställningen av produkten har en enkätundersökning gjorts som samtliga IISEE-instruktörer hade möjlighet att svara på. I kapitlet ”Processbeskrivning” redogörs för hur produkten utformats och vilka handlingar som gjorts inom respektive faser av produktutvecklingsprocessen.This degree thesis is a functional product development work issued by the Finnish company IISEE. IISEE is both an indoor cycling concept (spinning) and education organization for indoor cycling instructors. The company has been operating since 2008. In Finland, the number of trained IISEE instructors is about a 1000 of which approximately 200 are active today. The aim of this thesis is to develop a product (IISEE Tutor Community) based on tutoring, which operates as a coherent and systematic whole. The main task of the product is to increase the activity and interaction between IISEE instructors and serve all parties affected by the product. The questions at issues which this study aims to answer are the following: Which attributes do IISEE instructors regards as necessary for the product. Which attributes do IISEE instructors desire and covet? What is possible and realistic to implement and what is not regarding the product? How can the current system be utilized and developed with the launch of the new product? What does a well-functioning tutoring signify? How does the selection system work for the IISEE instructors who applies for the tutor vacancy? Can the tutors be rewarded for their work effort in a form other than money? The degree thesis consists of a theoretical part where general matters about indoor cycling, IISEE as a company and tutoring are described. Following this is the questions at issues and method. The method used in this work is process description and is based on the book Toiminnallinen opinnäytetyö (2003), written by Vilkka and Airaksinen. The product development process itself follows a five phase, step-by-step method of a product development which is based on the book Osaamisen tuotteistaminen sosiaali- ja terveysalalla (2000), written by Jämsä and Manninen. To support the development of the product a survey was made that all IISEE instructors had the opportunity to answer. In the chapter "Processbeskrivning", the product development process is thoroughly described regarding how the product was designed and the actions undertaken in the respective phases of the product development process.Tämä opinnäytetyö on toiminnalliseen tuotekehitykseen perustuva työ liittyen suomalaiseen yritykseen IISEE:hen. IISEE on sekä sisäpyöräilykonsepti (spinning) että koulutusorganisaatio sisäpyöräilyohjaajille. Yritys on toiminut vuodesta 2008 alkaen. Suomessa on koulutettu yhteensä noin 1000 IISEE-ohjaajaa, joista noin 200 on aktiivisia tällä hetkellä. Tämän opinnäytetyön tarkoituksena on kehittää tuote (IISEE Tutor Community), joka perustuu tutortoiminnalle ja toimii koherenttina ja systemaattisena kokonaisuutena. Tuotteen päätehtävänä on lisätä toimintaa ja vuorovaikutusta IISEE-ohjaajien välillä ja palvella kaikkia tuotteen osapuolia. Työn kysymyksenasettelu on seuraava: Millaiset ominaisuudet ovat IISEE-ohjaajien mielestä välttämättömiä tuotteelle? Mitä IISEE-ohjaajat toivovat ja haluavat tuotteelta? Mikä on realistista ja mahdollista toteuttaa ja mikä ei? Kuinka nykyistä järjestelmää voidaan hyödyntää ja kehittää tuotteen lanseerauksessa? Mitä hyvin toimiva tutortoiminta sisältää? Kuinka IISEE-ohjaaja voidaan valita tutoriksi? Voidaanko tutoreita palkita panoksestaan muutoin kuin rahan muodossa? Opinnäytetyö koostuu teoreettisesta osuudesta, jossa esitellään sisäpyöräily yleisesti, IISEE yrityksenä ja mitä tutortoiminta sisältää. Tämän jälkeen seuraa kysymyksen asettelu ja metodi. Tässä työssä on käytetty metodina prosessikirjoittamista, perustuen Vilkka ja Airaksisen kirjaan Toiminnallinen opinnäytetyö (2003). Itse tuotteenkehittämisprosessi seuraa tuotteenkehittämistyössä käytettyä viittä vaihetta, jotka perustuvat Jämsän ja Mannisen kirjaan Osaamisen tuotteistaminen sosiaali- ja terveysalalla (2000). Tuotteen kehittämisen tueksi on tehty kyselytutkimus, johon kaikilla IISEE-ohjaajilla on ollut mahdollisuus vastata. Kappaleessa ”Processbeskrivning” on perusteellisesti kuvattu, kuinka tuote on suunniteltu ja pohdittu, millaisia toimia tuotekehitysprosessin eri vaiheissa on toteutettu

2DDB – a bioinformatics solution for analysis of quantitative proteomics data

BACKGROUND: We present 2DDB, a bioinformatics solution for storage, integration and analysis of quantitative proteomics data. As the data complexity and the rate with which it is produced increases in the proteomics field, the need for flexible analysis software increases. RESULTS: 2DDB is based on a core data model describing fundamentals such as experiment description and identified proteins. The extended data models are built on top of the core data model to capture more specific aspects of the data. A number of public databases and bioinformatical tools have been integrated giving the user access to large amounts of relevant data. A statistical and graphical package, R, is used for statistical and graphical analysis. The current implementation handles quantitative data from 2D gel electrophoresis and multidimensional liquid chromatography/mass spectrometry experiments. CONCLUSION: The software has successfully been employed in a number of projects ranging from quantitative liquid-chromatography-mass spectrometry based analysis of transforming growth factor-beta stimulated fi-broblasts to 2D gel electrophoresis/mass spectrometry analysis of biopsies from human cervix. The software is available for download at SourceForge

Greedy de novo motif discovery to construct motif repositories for bacterial proteomes

BACKGROUND Bacterial surfaces are complex systems, constructed from membranes, peptidoglycan and, importantly, proteins. The proteins play crucial roles as critical regulators of how the bacterium interacts with and survive in its environment. A full catalog of the motifs in protein families and their relative conservation grade is a prerequisite to target the protein-protein interaction that bacterial surface protein makes to host proteins. RESULTS In this paper, we propose a greedy approach to identify conserved motifs in large sequence families iteratively. Each iteration discovers a motif de novo and masks all occurrences of that motif. Remaining unmasked sequences are subjected to the next round of motif detection until no more significant motifs can be found. We demonstrate the utility of the method through the construction of a proteome-wide motif repository for Group A Streptococcus (GAS), a significant human pathogen. GAS produce numerous surface proteins that interact with over 100 human plasma proteins, helping the bacteria to evade the host immune response. We used the repository to find that proteins part of the bacterial surface has motif architectures that differ from intracellular proteins. CONCLUSIONS We elucidate that the M protein, a coiled-coil homodimer that extends over 500 A from the cell wall, has a motif architecture that differs between various GAS strains. As the M protein is known to bind a variety of different plasma proteins, the results indicate that the different motif architectures are responsible for the quantitative differences of plasma proteins that various strains bind. The speed and applicability of the method enable its application to all major human pathogens

T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis

INTRODUCTION: A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model A(q) has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1(*)0401) and DR1 (DRB1(*)0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in A(q)-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the A(q)molecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules. Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4]. We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II A(q) molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants. OBJECTIVES: To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis. MATERIALS AND METHODS: A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG(1) and IgG(2a) levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 10(6) cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16]. T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (10(7)) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry. RESULTS AND DISCUSSION: To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days. To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG(1) versus IgG(2a) titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis. In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to A(q), it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25]

Quantitative proteogenomics of human pathogens using DIA-MS.

The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence

Identification of two abundant Aerococcus urinae cell wall-anchored proteins

Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6-24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease

Absolute quantification of microbial proteomes at different states by directed mass spectrometry

The developed, directed mass spectrometry workflow allows to generate consistent and system-wide quantitative maps of microbial proteomes in a single analysis. Application to the human pathogen L. interrogans revealed mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense, and new insights about the regulation of absolute protein abundances within operons

Comparative Functional Analysis of the Caenorhabditis elegans and Drosophila melanogaster Proteomes

The nematode Caenorhabditis elegans is a popular model system in genetics, not least because a majority of human disease genes are conserved in C. elegans. To generate a comprehensive inventory of its expressed proteome, we performed extensive shotgun proteomics and identified more than half of all predicted C. elegans proteins. This allowed us to confirm and extend genome annotations, characterize the role of operons in C. elegans, and semiquantitatively infer abundance levels for thousands of proteins. Furthermore, for the first time to our knowledge, we were able to compare two animal proteomes (C. elegans and Drosophila melanogaster). We found that the abundances of orthologous proteins in metazoans correlate remarkably well, better than protein abundance versus transcript abundance within each organism or transcript abundances across organisms; this suggests that changes in transcript abundance may have been partially offset during evolution by opposing changes in protein abundance