Pre-processing algorithms for Affymetrix microarrays

Zur genomweiten Genexpressionsanalyse werden Microarray-Experimente verwendet. Ziel dieser Arbeit ist es, Methoden zur Präprozessierung von Microarrays der Firma Affymetrix zu evaluieren und die VSN-Methode für Experimente mit weniger als 1000 Zellen zu verbessern. Bei dieser Technologie wird die Expression jedes Gens durch mehrere Probessets gemessen. Jedes Probeset besteht aus einem Perfect-Match (PM) und einem dazugehörigen Mismatch (MM). Der Expressionswert pro Gen wird durch ein vierstufiges Verfahren aus den einzelnen Probe-Werten berechnet: Hintergrundkorrektur, Normalisierung, PM-Adjustierung und Aggregation. Für jeden dieser Schritte existieren mehrere Algorithmen. Dazu dienten die im affy-Paket des Bioconductor implementierten Methoden MAS5, RMA, VSN und die Methode sRMA von Cope et al. [Cope et al., 2006] in Kombination mit der Methode VSN von Huber et al. [Huber et al., 2002]. Den ersten Teil dieser Arbeit bildet die Reanalyse der Datensätze von Küppers et al. [Küppers et al., 2003] und Piccaluga et al. [Piccaluga et al., 2007] mit der VSN-Methode. Dabei konnte gezeigt werden, dass die VSN-Methode gegenüber Klein et al. [Klein et al., 2001] Vorteile zeigt. Bei beiden Datensätzen wurden zusätzliche Gene gefunden, die für die Pathogenese der jeweiligen Tumorarten wichtig sein können. Einige der zusätzlich gefunden Gene wurden durch andere wissenschaftliche Arbeiten bestätigt. Die Gene, die bisher in keinem Zusammenhang mit der untersuchten Tumorart stehen, sind eine Möglichkeit für die weitere Forschung. Vor allem der Zytokine/Zytokine Signalweg wurde bei beiden Reanalysen als überrepräsentiert erkannt. Da für einige Microarray-Experimente die Anzahl der Zellen und damit die Menge an mRNA nur begrenzt zur Verfügung stehen, müssen die Laborarbeit und die statistischen Analysen angepasst werden. Hierzu werden fünf Methoden für die Präprozessierung untersucht, um zu evaluieren, welche Methode geeignet ist, derartige Expressionsdaten zu verrechnen. Auf Basis eines Testdatensatzes der bereits zur Etablierung des Laborprozesses diente werden Expressionswerte durch empirische Verteilung, Gammaverteilung und ein linear gemischtes Modell simuliert. Die Simulation lässt sich in vier Schritte einteilen: Wahl der Verteilung, Simulation der Expressionsmatrix, Simulation der differentiellen Expression, Sortierung der Probes innerhalb des Probesets. Anschließend werden die fünf Präprozessierungsmethoden mit diesen simulierten Expressionsdaten auf ihre Sensitivität und Spezifität untersucht. Während sich bei den empirisch und gammaverteilt simulierten Expressionsdaten kein eindeutiges Ergebnis abzeichnet, hat sVSN bei den Daten aus dem linear gemischten Modell die größte Sensitivität und die größte Spezifität. Der in dieser Arbeit entwickelte sVSN-Algorithmus wurde zum ersten Mal angewendet und bewertet. Abschließend wird ein Teildatensatz von Brune et al. verwendet und hinsichtlich der fünf Präprozessierungsmethoden untersucht. Die Ergebnisse der sVSN-Methode wird im Detail weiter verfolgt. Die zusätzlich gefunden Gene können durch bereits veröffentlichte Arbeiten bestätigt werden. Letztendlich zeigt sich, dass neuere statistische Methoden (wie das im Rahmen dieser Arbeit entwickelte sVSN) bei der Analyse von Affymetrix Microarrays einen Vorteil bringen. Die sVSN und sRMA Methoden zeigen Vorteile, da die Probes nach der Normalisierung gewichtet werden, bevor diese aggregiert werden. Die MAS5-Methode schneidet am schlechtesten ab und sollte bei geringen Zellmengen nicht eingesetzt werden. Für die Analyse mit geringer Menge an mRNA müssen weitere Untersuchungen vorgenommen werden, um eine geeignete statistische Methode für die Analyse der Expressionsdaten zu finden.Microarray experiments are used for genome-wide genetic expression analysis. An aim of this work is to evaluate methods of pre-processing Affymetrix microarrays and to improve the VSN method for experiments with less than 1000 cells. This technology measures the expression of genes by several probesets. Every probeset consists of a Perfect-Match (PM) and a corresponding Mismatch (MM). The expression value is calculated by a 4-step procedure from the probeset values: Background correction, normalisation, PM-adjustment and aggregation. For each of these steps several algorithms exist. The pre-processing algorithms MAS5, RMA, VSN and the method sRMA from Cope et al. [Cope et al., in 2006] in combination with the method of VSN from Huber et al. [Huber et al., in 2002] are chosen in further analysis. All of them are implemented in the affy package of the Bioconductor software. The first part of this work reanalyses the records of Küpper et al. [Küpper et al., in 2003] and Piccaluga et al. [Piccaluga et al., in 2007] with the VSN-method. It could be shown that the reanalysis with the VSN method is superior to the method Klein et al. [Klein et al., in 2001] used. Within both datasets additional promising genes were found that could be relevant for the pathogenesis of the respective tumour kinds. Moreover we revealed genes that were already confirmed by other scientific works meanwhile. Genes that are not known in connection with the examined tumour kind yet, might be interesting for further research. Mainly the Cytokine/Cytokine pathway showed up overrepresented in both reanalyses. Since in some microarray experiments the number of the cells and with it the amount of mRNA is limited, the lab work and the statistical analyses must be adapted. Moreover the five pre-processing methods are evaluated with regard to this special kind of expression data. On the basis of a test record - that was already used to establish the lab process – expression values were simulated by an empiric distribution, a gamma distribution and a linear mixed model. The simulation is divided in four steps: Choice of the distribution, simulation of the expression matrix, simulation of differential expression and sorting of the probes within the test set. Afterwards the five pre-processing methods are applied to the simulated expression data and examined for their sensitivity and specificity. Whereas the results for the expression data simulated with gamma-distribution and the empirical distribution are ambiguous, the sVSN method applied to the data from the linear mixed model shows the biggest sensitivity and specificity. The sVSN algorithm - that is introduced in this work - is applied for the first time and evaluated. Finally a partial record of Brune et al. is examined with these five pre-processing methods. The results of the sVSN method are inspected in more detail. Additional genes were found and some of them had already been confirmed by published works. To sum it up it is reasonable to apply more advanced statistical methods - like the sVSN method - in the analysis of Affymetrix microarrays. The sVSN and sRMA methods show advantages because the probes are weighted between the normalisation and aggregation steps. The MAS5-method is inferior compared to the others and should not be applied to expression data from low cell amounts. For the analysis of expression data coming from a small quantity of mRNA more research needs to be carried out to improve suitable statistical methods

GLUT1 expression patterns in different Hodgkin lymphoma subtypes and progressively transformed germinal centers

Background: Increased glycolytic activity is a hallmark of cancer, allowing staging and restaging with 18F-fluorodeoxyglucose-positron-emission-tomography (PET). Since interim-PET is an important prognostic tool in Hodgkin lymphoma (HL), the aim of this study was to investigate the expression of proteins involved in the regulation of glucose metabolism in the different HL subtypes and their impact on clinical outcome. Methods: Lymph node biopsies from 54 HL cases and reactive lymphoid tissue were stained for glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) and lactate exporter proteins MCT1 and MCT4. In a second series, samples from additional 153 HL cases with available clinical data were stained for GLUT1 and LDHA. Results: Membrane bound GLUT1 expression was frequently observed in the tumor cells of HL (49% of all cases) but showed a broad variety between the different Hodgkin lymphoma subtypes: Nodular sclerosing HL subtype displayed a membrane bound GLUT1 expression in the Hodgkin-and Reed-Sternberg cells in 56% of the cases. However, membrane bound GLUT1 expression was more rarely observed in tumor cells of lymphocyte rich classical HL subtype (30%) or nodular lymphocyte predominant HL subtype (15%). Interestingly, in both of these lymphocyte rich HL subtypes as well as in progressively transformed germinal centers, reactive B cells displayed strong expression of GLUT1. LDHA, acting downstream of glycolysis, was also expressed in 44% of all cases. We evaluated the prognostic value of different GLUT1 and LDHA expression patterns; however, no significant differences in progression free or overall survival were found between patients exhibiting different GLUT1 or LDHA expression patterns. There was no correlation between GLUT1 expression in HRS cells and PET standard uptake values. Conclusions: In a large number of cases, HRS cells in classical HL express high levels of GLUT1 and LDHA indicating glycolytic activity in the tumor cells. Although interim-PET is an important prognostic tool, a predictive value of GLUT1 or LDHA staining of the primary diagnostic biopsy could not be demonstrated. However, we observed GLUT1 expression in progressively transformed germinal centers and hyperplastic follicles, explaining false positive results in PET. Therefore, PET findings suggestive of HL relapse should always be confirmed by histology

Contraception in the German-language Wikipedia: a content and quality analysis

BACKGROUND: Adolescents and adults today often obtain information about contraception from the Internet, especially from the online encyclopedia Wikipedia. This is because Google searches usually return Wikipedia entries as top hits. RESEARCH AIM: Against this background, the aim of the current study is to systematically analyze for the first time the content and quality of Wikipedia articles on contraceptive methods. Five central quality dimensions are examined: the degree of correctness (research question RQ1), completeness (RQ2), neutrality (RQ3), comprehensibility (RQ4), and currency (RQ5) of the contraceptive information – and on this basis the overall quality of the articles (RQ6). MATERIALS AND METHODS: A sample of all German-language Wikipedia articles on all contraceptive methods was formed (N = 25). These articles were analyzed by three independent, trained coders using a codebook that was developed based on the current state of the research and tested for reliability. Data analysis was performed using SPSS. The study is preregistered and all data, materials, and analysis scripts are publicly available. RESULTS: The 25 Wikipedia articles on contraceptive methods were found to vary widely in content quality. While they showed good quality on average in terms of correctness (RQ1) and neutrality (RQ3), they scored mediocre in terms of completeness (RQ2), comprehensibility (RQ4), and currency (RQ5), resulting in moderate overall quality (RQ6). DISCUSSION: More research as well as practice measures are needed to further assess and improve the quality of contraceptive information on Wikipedia and in other social media in a more targeted way

Frequency of impulse control behaviours associated with dopaminergic therapy in restless legs syndrome.

BACKGROUND: Low doses of dopamine agonists (DA) and levodopa are effective in the treatment of restless legs syndrome (RLS). A range of impulse control and compulsive behaviours (ICBs) have been reported following the use of DAs and levodopa in patients with Parkinson's disease. With this study we sought to assess the cross-sectional prevalence of impulse control behaviours (ICBs) in restless legs syndrome (RLS) and to determine factors associated with ICBs in a population cohort in Germany. METHODS: Several questionnaires based on validated and previously used instruments for assessment of ICBs were mailed out to patients being treated for RLS. Final diagnoses of ICBs were based on stringent diagnostic criteria after psychiatric interviews were performed. RESULTS: 10/140 RLS patients of a clinical cohort (7.1%) were finally diagnosed with ICBs, 8 of 10 on dopamine agonist (DA) therapy, 2 of 10 on levodopa. 8 of the 10 affected patients showed more than one type of abnormal behaviour. Among those who responded to the questionnaires 6/140 [4.3%] revealed binge eating, 5/140 [3.6%] compulsive shopping, 3/140 [2.1%] pathological gambling, 3/140 [2.1%] punding, and 2/140 [1.4%] hypersexuality in psychiatric assessments. Among those who did not respond to questionnaires, 32 were randomly selected and interviewed: only 1 patient showed positive criteria of ICBs with compulsive shopping and binge eating. ICBs were associated with higher DA dose (p = 0.001), younger RLS onset (p = 0.04), history of experimental drug use (p = 0.002), female gender (p = 0.04) and a family history of gambling disorders (p = 0.02), which accounted for 52% of the risk variance. CONCLUSION: RLS patients treated with dopaminergic agents and dopamine agonists in particular, should be forewarned of potential side effects. A careful history of risk factors should be taken.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Communicating the risk of MRSA: the role of clinical practice, regulation and other policies in five European countries

Background: The threat posed by Meticillin-resistant Staphylococcus aureus (MRSA) has taken on an increasingly pan-European dimension. This article aims to provide an overview of the different approaches to the control of MRSA adopted in five European countries (Austria, Germany, Netherlands, Spain, and the UK) and discusses data and reporting mechanisms, regulations, guidelines, and health policy approaches with a focus on risk communication. Our hypothesis is that current infection control practices in different European countries are implicit messages that contribute to the health-related risk communication and subsequently to the public perception of risk posed by MRSA. A reporting template was used to systematically collect information from each country. Discussion: Large variation in approaches was observed between countries. However, there were a number of consistent themes relevant to the communication of key information regarding MRSA, including misleading messages, inconsistencies in content and application of published guidelines, and frictions between the official communication and their adoption on provider level. Summary: The variability of recommendations within, and across, countries could be contributing to the perception of inconsistency. Having inconsistent guidelines and practices in place may also be affecting the level at which recommended behaviors are adopted. The discrepancy between the official, explicit health messages around MRSA and the implicit messages stemming from the performance of infection control measures should, therefore, be a key target for those wishing to improve risk communication

Nodular lymphocyte predominant hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma : endpoints of a spectrum of one disease?

In contrast to the commonly indolent clinical behavior of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), T cell/histiocyte rich large B cell lymphoma (THRLBCL) is frequently diagnosed in advanced clinical stages and has a poor prognosis. Besides the different clinical presentations of these lymphoma entities, there are variants of NLPHL with considerable histopathologic overlap compared to THRLBCL. Especially THRLBCL-like NLPHL, a diffuse form of NLPHL, often presents a histopathologic pattern similar to THRLBCL, suggesting a close relationship between both lymphoma entities. To corroborate this hypothesis, we performed gene expression profiling of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. In unsupervised analyses, the lymphomas did not cluster according to their entity. Moreover, even in supervised analyses, very few consistently differentially expressed transcripts were found, and for these genes the extent of differential expression was only moderate. Hence, there are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. Based on the gene expression studies, we identified BAT3/BAG6, HIGD1A, and FAT10/UBD as immunohistochemical markers expressed in the tumor cells of all three lymphomas. Characterization of the tumor microenvironment for infiltrating T cells and histiocytes revealed significant differences in the cellular composition between typical NLPHL and THRLBCL cases. However, THRLBCL-like NLPHL presented a histopathologic pattern more related to THRLBCL than NLPHL. In conclusion, NLPHL and THRLBCL may represent a spectrum of the same disease. The different clinical behavior of these lymphomas may be strongly influenced by differences in the lymphoma microenvironment, possibly related to the immune status of the patient at the timepoint of diagnosis

Origin and pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma as revealed by global gene expression analysis

The pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly because of the technical challenge of analyzing its rare neoplastic lymphocytic and histiocytic (L&H) cells, which are dispersed in an abundant nonneoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected L&H lymphoma cells in comparison to normal and other malignant B cells that indicated a relationship of L&H cells to and/or that they originate from germinal center B cells at the transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell–rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype, and deregulation of many apoptosis regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive nuclear factor {kappa}B activity and aberrant extracellular signal-regulated kinase signaling. Thus, these findings shed new light on the nature of L&H cells, reveal several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies

Whole exome sequencing of microdissected splenic marginal zone lymphoma: a study to discover novel tumor-specific mutations

BACKGROUND: Splenic marginal zone lymphoma (SMZL) is an indolent B-cell non-Hodgkin lymphoma and represents the most common primary malignancy of the spleen. Its precise molecular pathogenesis is still unknown and specific molecular markers for diagnosis or possible targets for causal therapies are lacking. METHODS: We performed whole exome sequencing (WES) and copy number analysis from laser-microdissected tumor cells of two primary SMZL discovery cases. Selected somatic single nucleotide variants (SNVs) were analyzed using pyrosequencing and Sanger sequencing in an independent validation cohort. RESULTS: Overall, 25 nonsynonymous somatic SNVs were identified, including known mutations in the NOTCH2 and MYD88 genes. Twenty-three of the mutations have not been associated with SMZL before. Many of these seem to be subclonal. Screening of 24 additional SMZL for mutations at the same positions found mutated in the WES approach revealed no recurrence of mutations for ZNF608 and PDE10A, whereas the MYD88 L265P missense mutation was identified in 15 % of cases. An analysis of the NOTCH2 PEST domain and the whole coding region of the transcription factor SMYD1 in eight cases identified no additional case with a NOTCH2 mutation, but two additional cases with SMYD1 alterations. CONCLUSIONS: In this first WES approach from microdissected SMZL tissue we confirmed known mutations and discovered new somatic variants. Recurrence of MYD88 mutations in SMZL was validated, but NOTCH2 PEST domain mutations were relatively rare (10 % of cases). Recurrent mutations in the transcription factor SMYD1 have not been described in SMZL before and warrant further investigatio

10 Jahre Bildungsstandards - Tagungsband des AK Grundschule in der GDM 2014

In diesem Tagungsband sind die Ergebnisse der Herbsttagung des Arbeitskreises Grundschule in der Gesellschaft für Didaktik der Mathematik (GDM) in Tabarz vom 07. bis 09. November 2014 zum Thema „10 Jahre Bildungsstandards – Rückblick und Perspektiven“ dokumentiert. Zehn Jahre nach der Verabschiedung der Bildungsstandards nutzte der Arbeitskreis die Tagung, um aus dem Blickwinkel der Grundschule eine Rückschau vorzunehmen und dabei mögliche Perspektiven herauszuarbeiten. Das Rahmenthema wurde in vier Hauptvorträgen im Plenum diskutiert. Zusätzlich wurde eine Podiumsdiskussion zum Thema durchgeführt. Wie es bereits lange Tradition ist, setzten sich außerdem Arbeitsgruppen zu den klassischen Themenfeldern Arithmetik, Geometrie, Sachrechnen und Daten, Zufall & Wahrscheinlichkeit sowie zu den Bereichen Vorschulische Bildung, Lehren & Forschen mit Neuen Medien in der Primarstufe und Lehrerfortbildung intensiv mit aktuellen Forschungs- und Praxisfragen auseinander.The Proceedings of the 2014 Conference of the Research Group on Primary Mathematics Education (Arbeitskreis Grundschule in der GDM) focus on the 10th anniversary of the ‘Bildungsstandards’. Four invited talks as well as a panel discussion addressed the main theme in plenum. Additionally, workings groups on the research areas arithmetic, geometry, modelling, data & probability, as well as groups on teacher education, ICT, and last not least early mathematics education offered discussions on current research issues