Neue bioinformatische und statistische Methoden für die Analyse von Massenspektrometrie-basierten phosphoproteomischen Daten

In living cells, reversible protein phosphorylation events propagate signals caused by external stimuli from the plasma membrane to their intracellular destinations. Aberrations in these signaling cascades can lead to diseases such as cancer. To identify and quantify phosphorylation events on a large scale, mass spectrometry (MS) has become the predominant technology. The large amount of data generated by MS requires efficient, tailor-made computational tools in order to draw meaningful biological conclusions. In this work, four new methods for analyzing MS-based phosphoproteomic data are presented. The first method, called SubExtractor, combines phosphoproteomic data with protein network information to identify differentially regulated subnetworks. The method is based on a Bayesian probabilistic model that accounts for information about both differential regulation and network topology, combined with a genetic algorithm and rigorous significance testing. The second method, called MeanRank test, is a global one-sample location test, which is based on the mean ranks across replicates, and internally estimates and controls the false discovery rate. The test successfully deals with small numbers of replicates, missing values without the need of imputation, non-normally distributed expression levels, and non-identical distribution of up- and down-regulated features, while its statistical power scales well with the number of replicates. The third method is a biomarker discovery workflow that aims at identifying a multivariate response prediction biomarker for treatment of non-small cell lung cancer cell lines with the kinase inhibitor dasatinib from phosphoproteomic data (referred to as NSCLC biomarker). An elaborate biomarker workflow based on robust feature selection in combination with a support vector machine (SVM) was designed in order to find a phosphorylation signature that accurately predicts the response to dasatanib. The fourth method, called Pareto biomarker, extends the previous NSCLC biomarker workflow by optimizing not only one single objective (i.e. best possible separation of responders and non-responders), but also the objectives signature size and relevance (i.e. association of signature proteins with dasatinib’s main target). This is achieved by employing a multiobjective optimization algorithm based on the principle of Pareto optimality, which allows for a simultaneous optimization of all three objectives. These novel data analysis methods were thoroughly validated using experimental data and compared to existing methods. They can be used on their own, or they can be combined into a joint workflow in order to efficiently answer complex biological questions in the field of large-scale omics in general and phosphoproteomics in particular.In lebenden Zellen sind reversible Proteinphosphorylierungen für die Weiterleitung von Signalen externer Stimuli zu deren intrazellulären Bestimmungsorten verantwortlich. Anomalien in solchen Signaltransduktionswegen können zu Krankheiten wie beispielsweise Krebs führen. Um Phosphorylierungsstellen in großem Maßstab zu identifizieren und zu quantifizieren, hat sich die Massenspektrometrie (MS) zur vorherrschenden Technologie entwickelt. Die große Menge an Daten, die von Massenspektrometern generiert wird, erfordert effiziente maßgeschneiderte Computerprogramme, um aussagekräftige biologische Schlüsse ziehen zu können. In dieser Arbeit werden vier neue Methoden zur Analyse von MS-basierten phosphoproteomischen Daten präsentiert. Die erste Methode, genannt SubExtractor, kombiniert phosphoproteomische Daten mit Proteinnetzwerkinformationen um differentiell regulierte Subnetzwerke zu identifizieren. Die Methode basiert auf einem Bayesschen Wahrscheinlichkeitsmodell, das sowohl Information über die differentielle Regulation der Einzelknoten als auch die Netzwerktopologie berücksichtigt. Das Modell ist kombiniert mit einem genetischen Algorithmus und stringenter Signifikanzanalyse. Die zweite Methode, genannt MeanRank-Test, ist ein globaler Einstichproben-Lagetest, der auf den mittleren Rängen der Replikate beruht, und die False Discovery Rate implizit abschätzt und kontrolliert. Der Test eignet sich für die Anwendung auf Daten mit wenigen Replikate, fehlenden und nicht normalverteilten Werten, sowie nicht gleichverteilter Hoch- und Runterregulation. Gleichzeitig skaliert die Teststärke gut mit der Anzahl an Replikaten. Die dritte Methode ist ein Arbeitsablauf zur Biomarkeridentifizierung und hat zum Ziel, einen multivariaten Stratifikationsbiomarker aus phosphoproteomischen Daten zu extrahieren, der das Ansprechen von nichtkleinzelligen Bronchialkarzinomzelllinien auf den Kinaseinhibitor Dasatinib vorhersagt (bezeichnet als NSCLC-Biomarker). Dazu wurde ein ausführlicher Biomarkerarbeitsablauf basierend auf einer robusten Feature Selection in Kombination mit Support Vector Machine-Klassifizierung erstellt, um eine Phosphorylierungssignatur zu finden, die das Ansprechen auf Dasatinib richtig vorhersagt. Die vierte Methode, genannt Pareto-Biomarker, erweitert den vorherigen Biomarkerarbeitsablauf, indem nicht nur eine Zielfunktion (d.h. die bestmögliche Trennung von Respondern und Nichtrespondern) optimiert wird, sondern zusätzlich noch die Signaturgröße und Relevanz (d.h. die Verbindung der Signaturproteine mit dem Targetprotein von Dasatinib). Dies wird durch die Verwendung eines multiobjektiven Optimierungsalgorithmus erreicht, der auf dem Prinzip der Pareto-Optimalität beruht und die gleichzeitige Optimierung aller drei Zielfunktionen ermöglicht. Die hier präsentierten neuen Datenanalysemethoden wurden gründlich mittels experimenteller Daten validiert und mit bereits bestehenden Methoden verglichen. Sie können einzeln verwendet werden, oder man kann sie zu einem gemeinsamen Arbeitsablauf zusammenfügen, um komplexe biologische Fragestellungen in Omik-Gebieten im Allgemeinen und Phosphoproteomik im Speziellen zu beantworten

MetaTM - a consensus method for transmembrane protein topology prediction

Transmembrane (TM) proteins are proteins that span a biological membrane one or more times. As their 3-D structures are hard to determine, experiments focus on identifying their topology (i. e. which parts of the amino acid sequence are buried in the membrane and which are located on either side of the membrane), but only a few topologies are known. Consequently, various computational TM topology predictors have been developed, but their accuracies are far from perfect. The prediction quality can be improved by applying a consensus approach, which combines results of several predictors to yield a more reliable result. RESULTS: A novel TM consensus method, named MetaTM, is proposed in this work. MetaTM is based on support vector machine models and combines the results of six TM topology predictors and two signal peptide predictors. On a large data set comprising 1460 sequences of TM proteins with known topologies and 2362 globular protein sequences it correctly predicts 86.7% of all topologies. CONCLUSION: Combining several TM predictors in a consensus prediction framework improves overall accuracy compared to any of the individual methods. Our proposed SVM-based system also has higher accuracy than a previous consensus predictor. MetaTM is made available both as downloadable source code and as DAS server at http://MetaTM.sbc.su.se

Sulfate attack - Reaction mechanisms revealed by a multi proxy approach

The destructive effects of sulfate attack on concrete structures are well known, but the reaction paths and mechanisms that cause the deterioration are still under debate. The aim of this study is to contribute to a deeper understanding on investigating concrete damage by introducing a novel and promising multi proxy approach method. The methodology comprises advanced mineralogical and hydro-geochemical methods as well as stable isotope signals. Investigations were performed on various field case studies in Austria, where the locally occurring ground water was classified as slightly aggressive to concrete, in accordance to DIN EN 206-1. Nevertheless intense concrete damage related to sulfate attack was found. Severely damaged mushy concrete consisted mainly of thaumasite, secondary calcite, gypsum and relicts of aggregate. The expressed interstitial solutions from such material were extremely enriched in SO4 (up to >30000 mg L-1). Stable hydrogen and oxygen isotope were applied successfully and demonstrated that the degree of evaporation provoked enrichments in SO4 and other dissolved, potentially harmful ions such as Cl. Furthermore, the enormous accumulation of incompatible trace elements (e.g. Rb and Li) clearly indicated that numerous wetting and drying cycles had occurred. Such a highly dynamic system is known to induce severe destructive effects on concrete. In this study we demonstrate that the application of a multi proxy approach can provide a better understanding of the complexity of reaction mechanisms involving sulfate attack on concrete structures. More detailed knowledge on the individual reactions that promote concrete damage in field structures will help to find specific counter measures for already affected buildings and to develop tailored concrete recipes, applications and constructive measures for future projects

Use and acceptance of drinking fountains : a pilot study in two secondary schools in Dortmund, Germany

(1) Background: Water drinking is essential to reduce obesity in children, but effective means for implementation remain controversial. Our study assesses students’ and teachers’ use of and attitudes towards drinking fountains in two urban secondary schools. (2) Methods: In a cross-sectional study, answers from students and teachers to a 28- and 19-item questionnaire, respectively, containing closed- and open-ended questions and short interviews with the schools’ two principals were described and analysed using the question-specific number of responses as the denominator. (3) Results: Questionnaires of one hundred sixty-two students and ten teachers were analysed; 36.1% of students responded. Students viewed the schools’ two fountains as a good idea (73.3%, n = 118), recommended them to other schools (73.1%, n = 117), and felt able to distinguish healthy from unhealthy drinks (70.5%, n = 110). In contrast, 55.7% (n = 88) reported using the fountains regularly; over a week, 39.8% (n = 47) used them less than once; 26.3% (n = 31) used them one to two times. Only about a third (26.5%, n = 43) reported consuming more water since the fountains’ installation. Teachers’ responses were similar to students’; principals stressed planning and costs. (4) Conclusions: A discrepancy between a good attitude towards and actual use of drinking fountains may exist; school communities may need to look for measures to overcome it

Multicenter evaluation of blood-based biomarkers for the detection of endometriosis and adenomyosis: A prospective non-interventional study.

OBJECTIVE To evaluate blood-based biomarkers to detect endometriosis and/or adenomyosis across nine European centers (June 2014-April 2018). METHODS This prospective, non-interventional study assessed the diagnostic accuracy of 54 blood-based biomarker immunoassays in samples from 919 women (aged 18-45 years) with suspicion of endometriosis and/or adenomyosis versus symptomatic controls. Endometriosis was stratified by revised American Society for Reproductive Medicine stage. Symptomatic controls were "pathologic symptomatic controls" or "pathology-free symptomatic controls". The main outcome measure was receiver operating characteristic-area under the curve (ROC-AUC) and Wilcoxon P values corrected for multiple testing (q values). RESULTS CA-125 performed best in "all endometriosis cases" versus "all symptomatic controls" (AUC 0.645, 95% confidence interval [CI] 0.600-0.690, q < 0.001) and increased (P < 0.001) with disease stage. In "all endometriosis cases" versus "pathology-free symptomatic controls", S100-A12 performed best (AUC 0.692, 95% CI 0.614-0.769, q = 0.001) followed by CA-125 (AUC 0.649, 95% CI 0.569-0.729, q = 0.021). In "adenomyosis only cases" versus "symptomatic controls" or "pathology-free symptomatic controls", respectively, the top-performing biomarkers were sFRP-4 (AUC 0.615, 95% CI 0.551-0.678, q = 0.045) and S100-A12 (AUC 0.701, 95% CI 0.611-0.792, q = 0.004). CONCLUSION This study concluded that no biomarkers tested could diagnose or rule out endometriosis/adenomyosis with high certainty

Proteomics Reveals Novel Drosophila Seminal Fluid Proteins Transferred at Mating

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction

Topics in Noncommutative Geometry Inspired Physics

In this review article we discuss some of the applications of noncommutative geometry in physics that are of recent interest, such as noncommutative many-body systems, noncommutative extension of Special Theory of Relativity kinematics, twisted gauge theories and noncommutative gravity.Comment: New references added, Published online in Foundations of Physic