Within the fold: assessing differential expression measures and reproducibility in microarray assays

BACKGROUND: 'Fold-change' cutoffs have been widely used in microarray assays to identify genes that are differentially expressed between query and reference samples. More accurate measures of differential expression and effective data-normalization strategies are required to identify high-confidence sets of genes with biologically meaningful changes in transcription. Further, the analysis of a large number of expression profiles is facilitated by a common reference sample, the construction of which must be carefully addressed. RESULTS: We carried out a series of 'self-self' hybridizations in which aliquots of the same RNA sample were labeled separately with Cy3 and Cy5 fluorescent dyes and co-hybridized to the same microarray. From this, we can analyze the intensity-dependent behavior of microarray data, define a statistically significant measure of differential expression that exploits the structure of the fluorescent signals, and measure the inherent reproducibility of the technique. We also devised a simple procedure for identifying and eliminating low-quality data for replicates within and between slides. We examine the properties required of a universal reference RNA sample and show how pooling a small number of samples with a diverse representation of expressed genes can outperform more complex mixtures as a reference sample. CONCLUSION: Analysis of cell-line samples can identify systematic structure in measured gene-expression levels. A general procedure for analyzing cDNA microarray data is proposed and validated. We show that pooled reference samples should be based not only on the expression of individual genes in each cell line but also on the expression levels of genes within cell lines

Historical ‘signposts’ and other temporal indicators in the Czech lexicon

This article posits that the Czechs employ a great many historical markers, previously applied to other events of national importance, which help to shape collective memory and right the ‘wrongs’ of the past. It is argued that these temporal indicators share a number of clearly defined characteristics, and that their use is too systematic and calculated to be merely a function of the constraints of the lexicon. The first part of the study considers in detail questions of semantics (especially the distinction between denotation and connotation), the lexicographical sources available to the researcher, and the lexical ‘signpost’ in context, while the second part focuses on practical examples of lexical re-appropriation since 1918, with particular reference to dictionaries and the Czech National Corpus.University of Wolverhampto

Rare variants in single-minded 1 (SIM1) are associated with severe obesity

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.Shwetha Ramachandrappa, Anne Raimondo, Anna M.G. Cali, Julia M. Keough, Elana Henning, Sadia Saeed, Amanda Thompson, Sumedha Garg, Elena G. Bochukova, Soren Brage, Victoria Trowse, Eleanor Wheeler, Adrienne E. Sullivan, Mehul Dattani, Peter E. Clayton, Vippan Datta, John B. Bruning, Nick J. Wareham, Stephen O'Rahilly, Daniel J. Peet, Ines Barroso, Murray L. Whielaw and I. Sadaf Farooq

Variability and Diversity of Nasopharyngeal Microbiota in Children: A Metagenomic Analysis

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5–V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1000000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection

The Science of Pronominal Usage: He and It in Co-Reference to Inanimate Objects in Late Middle English Texts on Alchemy

This is the author's accepted manuscript. The published version can be found at http://dx.doi.org/10.1177/0075424210384225This article explores the variation between he and it in coreference to inanimate entities (such as mercury, sulfur, and salt). Using alchemical texts from the fifteenth century as material, the article demonstrates that there was much more variation in pronominal reference in this period than has previously been shown. Of the possible explanations suggested by previous research, the earlier grammatical gender system and transference from Latin do not seem to play a role, while pronoun clustering and pronominal reanalysis appear to influence the quantitative distribution. The scale of individuation used by Siemund and Stenroos to explain similar usage is shown not to be a straightforward predictor. Other factors such as personification and perceived similarities between animate and inanimate entities may affect the degree of perceived individuation. The choice of he over she seems to be influenced by pronominal reanalysis and straightforward personification in some cases. In other instances, it is speculated that the he usage reflects (stereotypical) gender conceptions in the Middle Ages

Human Sclera Maintains Common Characteristics with Cartilage throughout Evolution

BACKGROUND: The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. METHODOLOGY/PRINCIPAL FINDINGS: We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-betas and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human 'sclera'-derived cells can be considered chondrocytes when cultured ex vivo. CONCLUSIONS/SIGNIFICANCE: Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia

Measurement of the quasi-elastic axial vector mass in neutrino-oxygen interactions

The weak nucleon axial-vector form factor for quasi-elastic interactions is determined using neutrino interaction data from the K2K Scintillating Fiber detector in the neutrino beam at KEK. More than 12,000 events are analyzed, of which half are charged-current quasi-elastic interactions nu-mu n to mu- p occurring primarily in oxygen nuclei. We use a relativistic Fermi gas model for oxygen and assume the form factor is approximately a dipole with one parameter, the axial vector mass M_A, and fit to the shape of the distribution of the square of the momentum transfer from the nucleon to the nucleus. Our best fit result for M_A = 1.20 \pm 0.12 GeV. Furthermore, this analysis includes updated vector form factors from recent electron scattering experiments and a discussion of the effects of the nucleon momentum on the shape of the fitted distributions.Comment: 14 pages, 10 figures, 6 table

Measurement of the Branching Fraction for B- --> D0 K*-

We present a measurement of the branching fraction for the decay B- --> D0 K*- using a sample of approximately 86 million BBbar pairs collected by the BaBar detector from e+e- collisions near the Y(4S) resonance. The D0 is detected through its decays to K- pi+, K- pi+ pi0 and K- pi+ pi- pi+, and the K*- through its decay to K0S pi-. We measure the branching fraction to be B.F.(B- --> D0 K*-)= (6.3 +/- 0.7(stat.) +/- 0.5(syst.)) x 10^{-4}.Comment: 7 pages, 1 postscript figure, submitted to Phys. Rev. D (Rapid Communications

The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

Mass spectrometry (MS) based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS) data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007) described a modified spectral counting technique, Absolute Protein Expression (APEX), which improves on basic spectral counting methods by including a correction factor for each protein (called O(i) value) that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (O(i)). This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results: The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. Conclusion: The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.National Institute of Allergy and Infectious Diseases (NIAID) N01-AI15447National Institutes of HealthNational Science Foundation, the Welsh and Packard FoundationsInternational Human Frontier Science ProgramCenter for Systems and Synthetic Biolog

Study of e+e- --> pi+ pi- pi0 process using initial state radiation with BABAR

The process e+e- --> pi+ pi- pi0 gamma has been studied at a center-of-mass energy near the Y(4S) resonance using a 89.3 fb-1 data sample collected with the BaBar detector at the PEP-II collider. From the measured 3pi mass spectrum we have obtained the products of branching fractions for the omega and phi mesons, B(omega --> e+e-)B(omega --> 3pi)=(6.70 +/- 0.06 +/- 0.27)10-5 and B(phi --> e+e-)B(phi --> 3pi)=(4.30 +/- 0.08 +/- 0.21)10-5, and evaluated the e+e- --> pi+ pi- pi0 cross section for the e+e- center-of-mass energy range 1.05 to 3.00 GeV. About 900 e+e- --> J/psi gamma --> pi+ pi- pi0 gamma events have been selected and the branching fraction B(J/psi --> pi+ pi- pi0)=(2.18 +/- 0.19)% has been measured.Comment: 21 pages, 37 postscript figues, submitted to Phys. Rev.