Analysis of a simulated microarray dataset: Comparison of methods for data normalisation and detection of differential expression (Open Access publication)

Microarrays allow researchers to measure the expression of thousands of genes in a single experiment. Before statistical comparisons can be made, the data must be assessed for quality and normalisation procedures must be applied, of which many have been proposed. Methods of comparing the normalised data are also abundant, and no clear consensus has yet been reached. The purpose of this paper was to compare those methods used by the EADGENE network on a very noisy simulated data set. With the a priori knowledge of which genes are differentially expressed, it is possible to compare the success of each approach quantitatively. Use of an intensity-dependent normalisation procedure was common, as was correction for multiple testing. Most variety in performance resulted from differing approaches to data quality and the use of different statistical tests. Very few of the methods used any kind of background correction. A number of approaches achieved a success rate of 95% or above, with relatively small numbers of false positives and negatives. Applying stringent spot selection criteria and elimination of data did not improve the false positive rate and greatly increased the false negative rate. However, most approaches performed well, and it is encouraging that widely available techniques can achieve such good results on a very noisy data set.     

Heterologous expression of flowering locus T promotes flowering but does not affect diurnal movement in the legume Lotus japonicus

Flowering locus T (FT) is known to promote flowering in response to photoperiodic conditions and has recently been shown to contribute to other phenomenon, such as diurnal stomatal movement. In legumes, FTs are classified into three subtypes, though the role of each subtype is not well defined. It has been reported that when FT of Lotus japonicus (LjFT) is heterologously expressed in Arabidopsis, LjFT functions as a mobile florigen to promote flowering, similar to Arabidopsis FT (AtFT). In this study, we expressed AtFT in L. japonicus using the SUC2 promoter and showed that heterologous expression of AtFT was able to promote flowering in the plant. We also showed that AtFT expression does not affect stomatal closing nor nyctinastic leaf movement. These findings contribute to our understanding of flower development and have potential application to breeding or plant biotechnology.     

The Effects of Twitter Sentiment on Stock Price Returns

Social media are increasingly reflecting and influencing behavior of other complex systems. In this paper we investigate the relations between a well-known micro-blogging platform Twitter and financial markets. In particular, we consider, in a period of 15 months, the Twitter volume and sentiment about the 30 stock companies that form the Dow Jones Industrial Average (DJIA) index. We find a relatively low Pearson correlation and Granger causality between the corresponding time series over the entire time period. However, we find a significant dependence between the Twitter sentiment and abnormal returns during the peaks of Twitter volume. This is valid not only for the expected Twitter volume peaks (e.g., quarterly announcements), but also for peaks corresponding to less obvious events. We formalize the procedure by adapting the well-known “event study” from economics and finance to the analysis of Twitter data. The procedure allows to automatically identify events as Twitter volume peaks, to compute the prevailing sentiment (positive or negative) expressed in tweets at these peaks, and finally to apply the “event study” methodology to relate them to stock returns. We show that sentiment polarity of Twitter peaks implies the direction of cumulative abnormal returns. The amount of cumulative abnormal returns is relatively low (about 1–2%), but the dependence is statistically significant for several days after the events.     

Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis lectin (CNL), the N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc)-specific lectin from basidiomycete C. nebularis

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.     

Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein

Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.     

Montan wax improves performance of boron-based wood preservatives

Importance of boron compounds in wood preservation is increasing due to their low environmental impact, high efficacy and the fact that many other active ingredients have been removed from the market after the introduction of the Biocidal Products Directive. The most important drawback of boron is prominent leaching in wet environment. In order to improve their fixation, and performance against wood decay fungi, boric acid was combined with montan wax emulsion. Possible synergistic effects of boric acid and montan wax were determined according to modified EN 113 procedure. Norway spruce and beech wood specimens were exposed to three white rot (Trametes versicolor, Pleurotus ostreatus and Hypoxylon fragiforme) and brown rot wood decay fungi (Gloeophyllum trabeum, Antrodia vaillantii and Serpula lacrymans) for 12 weeks. Boron leaching from vacuum/pressure treated Norway spruce wood was determined according to the continuous (EN 84 and ENV 1250-2) and non-continuous (OECD and prCEN/TS 15119-1) procedures. Boron was determined with ICP mass spectrometry in collected leachates. The results of the fungicidal tests clearly showed that montan wax emulsion and boric acid act synergistically against tested wood decay fungi. Approximately 50% lower boric acid retentions are required in combination with montan wax emulsions to achieve sufficient protection against wood rotting fungi. However, it is even more important that all leaching tests performed proved that the addition of montan wax decreased boron leaching from impregnated specimens for 20% up to 50%.     

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.     

Antibody variable-region sequencing as a method for hybridoma cell-line authentication

Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Simon Koren and Miha Kosmač contributed equally to this work.     

Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells

Testican-2 is a member of the testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was approximately 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit cathepsins L, B, K and S. We identified the C1q subcomponent of complement component C1 as a potential interacting partner of testican-2. The C1q subcomponent is a recognition molecule which acts in concert with other C1 subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes.