Density based pruning for identification of differentially expressed genes from microarray data

<p>Abstract</p> <p>Motivation</p> <p>Identification of differentially expressed genes from microarray datasets is one of the most important analyses for microarray data mining. Popular algorithms such as statistical t-test rank genes based on a single statistics. The false positive rate of these methods can be improved by considering other features of differentially expressed genes.</p> <p>Results</p> <p>We proposed a pattern recognition strategy for identifying differentially expressed genes. Genes are mapped to a two dimension feature space composed of average difference of gene expression and average expression levels. A density based pruning algorithm (DB Pruning) is developed to screen out potential differentially expressed genes usually located in the sparse boundary region. Biases of popular algorithms for identifying differentially expressed genes are visually characterized. Experiments on 17 datasets from Gene Omnibus Database (GEO) with experimentally verified differentially expressed genes showed that DB pruning can significantly improve the prediction accuracy of popular identification algorithms such as t-test, rank product, and fold change.</p> <p>Conclusions</p> <p>Density based pruning of non-differentially expressed genes is an effective method for enhancing statistical testing based algorithms for identifying differentially expressed genes. It improves t-test, rank product, and fold change by 11% to 50% in the numbers of identified true differentially expressed genes. The source code of DB pruning is freely available on our website <url>http://mleg.cse.sc.edu/degprune</url></p

Gene Expression Signatures Can Aid Diagnosis of Sexually Transmitted Infection-Induced Endometritis in Women

Sexually transmitted infection (STI) of the upper reproductive tract can result in inflammation and infertility. A biomarker of STI-induced upper tract inflammation would be significant as many women are asymptomatic and delayed treatment increases risk of sequelae. Blood mRNA from 111 women from three cohorts was profiled using microarray. Unsupervised analysis revealed a transcriptional profile that distinguished 9 cases of STI-induced endometritis from 18 with cervical STI or uninfected controls. Using a hybrid feature selection algorithm we identified 21 genes that yielded maximal classification accuracy within our training dataset. Predictive accuracy was evaluated using an independent testing dataset of 5 cases and 10 controls. Sensitivity was evaluated in a separate test set of 12 women with asymptomatic STI-induced endometritis in whom cervical burden was determined by PCR; and specificity in an additional test set of 15 uninfected women with pelvic pain due to unknown cause. Disease module preservation was assessed in 42 women with a clinical diagnosis of pelvic inflammatory disease (PID). We also tested the ability of the biomarker to discriminate STI-induced endometritis from other diseases. The biomarker was 86.7% (13/15) accurate in correctly distinguishing cases from controls in the testing dataset. Sensitivity was 83.3% (5/6) in women with high cervical Chlamydia trachomatis burden and asymptomatic endometritis, but 0% (0/6) in women with low burden. Specificity in patients with non-STI-induced pelvic pain was 86.7% (13/15). Disease modules were preserved in all 8 biomarker predicted cases. The 21-gene biomarker was highly discriminatory for systemic infections, lupus, and appendicitis, but wrongly predicted tuberculosis as STI-induced endometritis in 52.4%. A 21-gene biomarker can identify asymptomatic women with STI-induced endometritis that places them at risk for chronic disease development and discriminate STI-induced endometritis from non-STI pelvic pain and other diseases

The early dendritic cell signaling induced by virulent Francisella tularensis strain occurs in phases and involves the activation of ERKs and p38 in the later stage

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. While reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by Francisella tularensis. Presented results show that the early response of DCs to Francisella occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated ΔdsbA strain. These findings imply that the temporal orchestration of host pro-inflammatory pathways represents the integral part of Francisella life-cycle inside hijacked DCs

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

The effect of dietary omega-3 and -6 polyunsaturated fatty acids on ovine ovarian function and the pre-implantation embryo

There is considerable interest in the beneficial role of dietary polyunsaturated fatty acids (PUFA) on reproduction in ruminants. Detailed information regarding the mechanisms behind this beneficial effect is limited. The main objective of this thesis was to test the effects of dietary supplementation with omega-3 (n-3) or -6 (n-6) PUFA on gene expression, fatty acid (FA) composition and steroidogenesis in granulosa and theca cells and pre-implantation embryo development. A previous study in our laboratory reported increased follicular-fluid progesterone concentrations in ewes fed an n-3 compared to an n-6 PUFA-enriched diet, but detected no differential effect of n-3 and n-6 PUFA enriched high-density lipoproteins (HDL) on granulosa cell steroidogenesis in vitro. Also, n-6 PUFA enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. In view of these observations it was hypothesised that (i) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than granulosa cells and (ii) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin delivered n- 3 and n-6 PUFA would exert a greater differential effect on embryo development than either LDL or HDL delivered PUFA. Initial investigations into granulosa cell gene expression profiles using an ovine gonad-targeted cDNA macroarray were unsuccessful, highlighted by subsequent qRTPCR analysis. A thorough investigation confirmed that inconsistencies were due to poor array hybridisation. In vitro data confirmed that n-3 PUFA, via delivery by HDL, increase progesterone production solely in theca cells and that this is associated with an increase in STAR transcript expression. We also demonstrate that albumin is the only serum fraction that leads to a net uptake of FA during embryo culture. PUFA enriched serum and albumin accelerated the development of embryos and increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differentially effects of PUFA on embryo development are less apparent

Proteomická analýza leukemických buněčných linií po ozáření

(CZE) Název dizertační práce: Proteomická analýza leukemických buněčných linií po ozáření V této dizertační práci jsme se zaměřili na objasnění molekulárních mechanismů radiosensibilizace leukemické buněčné linie MOLT-4 specifickou inhibicí kináz z rodiny fosfatidylinositol-3-kináza příbuzných kináz (PIKKs). Byly testovány dva vysoce účinné a selektivní inhibitory VE-821 (inhibitor ATR) a KU55933 (inhibitor ATM), pro jejich účinky na proliferaci, viabilitu a buněčný cyklus neozářených a ozářených buněk MOLT4. Aplikace obou inhibitorů způsobila radiosensibilizaci MOLT-4 buněk a 10 µM VE-821 navíc působil jako silné antiproliferativní agens i v neozářených MOLT-4 buňkách. K dalšímu popisu mechanismů, které jsou zodpovědné za radiosensibilizaci MOLT-4 buněk VE-821 inhibitorem byly použity hmotnostně spektrometrické metody. Pomocí metod kvantitativní proteomiky jsme identifikovali a kvantifikovali změny v proteomu a fosfoproteomu (tj. změny na úrovni fosforylace proteinů) buněk, které byly způsobeny účinkem inhibitoru v ozářených buňkách. Protože detekce a kvantifikace fosforylovaných peptidů v komplexních vzorcích je komplikována mimo jiné jejich relativně nízkým zastoupením, zaměřili jsme se nejprve na výběr optimální metody pro jejich selektivní izolaci ze směsi s nemodifikovanými peptidy....(ENG) Title of the dissertation: Proteomic analysis of gamma-irradiated human leukemic cells In the presented doctoral thesis, we aimed to elucidate molecular mechanisms underlying radiosensitization of MOLT-4 cell line (T-ALL) by specific inhibition of kinases from the phosphatidylinositol-3 kinase-related kinases (PIKKs) family. We tested two highly potent inhibitors of ATR and ATM, VE-821 and KU55933, respectively, for their effects on proliferation, viability, and cell cycle of sham-irradiated and irradiated MOLT-4 cells. Both inhibitors proved to radiosensitize MOLT-4 cells and furthermore, 10 µM VE-821 was shown to act as a strong antiproliferative agent in sham-irradiated MOLT-4 cells. To further describe cellular mechanisms underlying the VE-821-mediated radiosensitization of MOLT-4 cells, we employed high-resolution mass spectrometry to identify and quantify changes in proteome and phosphoproteome of irradiated VE-821-treated cells. As the detection and quantification of phosphorylated peptides in complex biological samples is challenging due to their low stoichiometry, we first compiled and optimized protocol for their enrichment. The protocol was then applied to study changes in radiosensitized MOLT-4 cells. In concordance with our expectations, VE-821 did not cause any significant...Department of Medical BiochemistryÚstav lékařské biochemieFaculty of Medicine in Hradec KrálovéLékařská fakulta v Hradci Králov

The effect of dietary omega-3 and -6 polyunsaturated fatty acids on ovine ovarian function and the pre-implantation embryo

There is considerable interest in the beneficial role of dietary polyunsaturated fatty acids (PUFA) on reproduction in ruminants. Detailed information regarding the mechanisms behind this beneficial effect is limited. The main objective of this thesis was to test the effects of dietary supplementation with omega-3 (n-3) or -6 (n-6) PUFA on gene expression, fatty acid (FA) composition and steroidogenesis in granulosa and theca cells and pre-implantation embryo development. A previous study in our laboratory reported increased follicular-fluid progesterone concentrations in ewes fed an n-3 compared to an n-6 PUFA-enriched diet, but detected no differential effect of n-3 and n-6 PUFA enriched high-density lipoproteins (HDL) on granulosa cell steroidogenesis in vitro. Also, n-6 PUFA enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. In view of these observations it was hypothesised that (i) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than granulosa cells and (ii) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin delivered n- 3 and n-6 PUFA would exert a greater differential effect on embryo development than either LDL or HDL delivered PUFA. Initial investigations into granulosa cell gene expression profiles using an ovine gonad-targeted cDNA macroarray were unsuccessful, highlighted by subsequent qRTPCR analysis. A thorough investigation confirmed that inconsistencies were due to poor array hybridisation. In vitro data confirmed that n-3 PUFA, via delivery by HDL, increase progesterone production solely in theca cells and that this is associated with an increase in STAR transcript expression. We also demonstrate that albumin is the only serum fraction that leads to a net uptake of FA during embryo culture. PUFA enriched serum and albumin accelerated the development of embryos and increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differentially effects of PUFA on embryo development are less apparent

B-Cell Receptor Signalling in Chronic Lymphocytic Leukaemia.

PhDChronic Lymphocytic Leukaemia (CLL) cells may depend on B-cell receptor (BCR) signalling as well as other microenvironmental survival signals. A peculiarity of CLL is that cells preserve IgD signalling in the presence of reduced IgM signalling, a pattern mimicking anergic B-cells, and consistent with autoantigen exposure. This aim of this thesis was to examine the differing roles of IgM and IgD in CLL. IgM and IgD expression was examined in CLL cells from peripheral blood (PB) and lymph node (LN). Co-expression of IgM and IgD was common, but levels of expression of IgD and IgM vary independently within these compartments, implying they may have differing roles. Most PB CLL samples underwent calcium (Ca) flux after IgD ligation, with evidence of relative IgM anergy. Incubation of cells for 24h in vitro partially restored IgM Ca flux, further supporting an anergic model. A pro-survival role of the BCR was suggested by the finding that BCR ligation was associated with reduced apoptosis in vitro. Mechanistic differences of IgM and IgD signalling were examined using mass-spectrometry based phosphoproteomics. Six CLL samples were compared to five tonsil controls, and >5000 unique phosphopeptides identified. CLL and tonsil phosphoproteomes were compared. BCR induced changes in phosphoproteins and kinases differed between IgM and IgD ligation, and between CLL and tonsillar B-cells. Recognised and novel pathways included BCR, spliceosome and Notch signalling. Evidence in support of anergic signalling in CLL was observed. Anergic IgM signalling is contrasted with IgD as a dynamic process, different in the tissue compartments of CLL. Phosphoproteomics offers a powerful tool for interrogating intracellular signalling, with phosphorylation networks characterizing pathway topology. BCR signalling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known as well as novel pathways that may well represent novel treatment targets.Cancer Research UK; Barts Cancer Institut