Correction to: Detect tissue heterogeneity in gene expression data with BioQC.

After the publication of this work [1], a mistake was noticed in the Eq. 1. Given an m × n expression matrix with m genes and samples of n tissues, the correct definition of the Gini index for gene i is.

Detect tissue heterogeneity in gene expression data with BioQC.

BACKGROUND: Gene expression data can be compromised by cells originating from other tissues than the target tissue of profiling. Failures in detecting such tissue heterogeneity have profound implications on data interpretation and reproducibility. A computational tool explicitly addressing the issue is warranted. RESULTS: We introduce BioQC, a R/Bioconductor software package to detect tissue heterogeneity in gene expression data. To this end BioQC implements a computationally efficient Wilcoxon-Mann-Whitney test and provides more than 150 signatures of tissue-enriched genes derived from large-scale transcriptomics studies. Simulation experiments show that BioQC is both fast and sensitive in detecting tissue heterogeneity. In a case study with whole-organ profiling data, BioQC predicted contamination events that are confirmed by quantitative RT-PCR. Applied to transcriptomics data of the Genotype-Tissue Expression (GTEx) project, BioQC reveals clustering of samples and suggests that some samples likely suffer from tissue heterogeneity. CONCLUSIONS: Our experience with gene expression data indicates a prevalence of tissue heterogeneity that often goes unnoticed. BioQC addresses the issue by integrating prior knowledge with a scalable algorithm. We propose BioQC as a first-line tool to ensure quality and reproducibility of gene expression data.

Genome-based analysis of the nonhuman primate Macaca fascicularis as a model for drug safety assessment.

The long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads. The sixfold sequence coverage of the draft genome sequence enabled discovery of about 2.1 million potential single-nucleotide polymorphisms based on occurrence of a dimorphic nucleotide at a given position in the genome sequence. Homology-based annotation allowed us to identify 17,387 orthologs of human protein-coding genes in the M. fascicularis draft genome, and the predicted transcripts enabled the design of a M. fascicularis-specific gene expression microarray. Using liver samples from 36 individuals of different geographic origin we identified 718 genes with highly variable expression in liver, whereas the majority of the transcriptome shows relatively stable and comparable expression. Knowledge of the M. fascicularis draft genome is an important contribution to both the use of this animal in disease models and the safety assessment of drugs and their metabolites. In particular, this information allows high-resolution genotyping and microarray-based gene-expression profiling for animal stratification, thereby allowing the use of well-characterized animals for safety testing. Finally, the genome sequence presented here is a significant contribution to the global "3R" animal welfare initiative, which has the goal to reduce, refine, and replace animal experiments.

Opposite effects of Zn on the in vitro binding of [3H]LY354740 to recombinant and native metabotropic glutamate 2 and 3 receptors.

We investigated the effect of Zn on agonist binding to both recombinant and native mGlu2 and mGlu3 receptors. Zn had a biphasic inhibitory effect on recombinant mGlu2 with IC(50) values for the high- and low-affinity components of 60 +/- 10 microM and 2 +/- 0.7 mM, respectively. Zn induced a complex biphasic effect of inhibition and enhancement of [(3)H]LY354740 binding to mGlu3. Observations with a series of chimeric mGlu2/3 receptors suggest that the Zn effect resides in the N-terminal domain of mGlu2 and mGlu3. We observed that the His56 of mGlu2, which corresponds to Asp63 in mGlu3 was largely accountable for the second phase of the Zn effect. As revealed by quantitative receptor radioautography, the addition of up to 100 microm Zn to brain sections of wild-type mice resulted in significant decreases in binding density in most brain regions. In particular, the mid-molecular layer of the dentate gyrus (DGmol) and the CA1 lacunosum moleculare of hippocampus (CA1-LMol) showed reductions of 62 and 67%, respectively. In contrast, the addition of 300 microM Zn to brain sections of mGlu2(-/-) mice caused large increases in binding density of 289 and 242% in DGmol and CA1-LMol, respectively. Therefore, Zn might play a role as a physiological modulator of group II mGlu receptor function.

Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket.

N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.

Expression modes of interferon-alpha inducible genes in sensitive and resistant human melanoma cells stimulated with regular and pegylated interferon-alpha.

Interferon-alpha with its antiproliferative activity is widely used for the treatment of viral infections and tumor therapy such as melanoma. Naturally occurring resistance to recombinant interferon alpha-2a (IFN-alpha) and severe side effects limit the therapeutic efficacy. Understanding of the molecular mechanisms involved in unresponsiveness may therefore lead to the development of novel formulations that overcome resistance. Here, we have applied oligonucleotide DNA microarrays with probe sets for about 11,400 human transcripts to study the expression of interferon-alpha inducible genes in a sensitive and resistant melanoma cell line over multiple time points and two interferon formulations. We identified two major groups of genes with termed interferon primary response genes (IPRGs) or interferon secondary response genes (ISRGs). IPRGs are upregulated early after interferon stimulation in both the sensitive and the resistant line and they contain IREs in the noncoding regulatory region. In contrast, ISRG expression occurs preferentially in the sensitive line ME15 at late time points, and this group of genes lacks typically IREs. In addition to these two major interferon response gene classes, we identified a relatively small number of genes with complex kinetic expression modes. In addition, we show for the first time that regular and pegylated recombinant interferons are equally potent activators of interferon (IFN) gene expression. Finally, we propose that the ISRGs are activated downstream of the primary response genes by a molecule or pathway, which awaits identification, and interferon inducible gene expression is thus more complicated than previously thought.

Statistical methods for analyzing microarray feature data with replications.

Expression levels in oligonucleotide microarray experiments depend on a potentially large number of factors, for example, treatment conditions, different probes, different arrays, and so on. To dissect the effects of these factors on expression levels, fixed-effects ANOVA methods have previously been proposed. Because we are not necessarily interested in estimating the specific effects of different probes and arrays, we propose to treat these as random effects. Then we only need to estimate their means and variances but not the effect of each of their levels; that is, we can work with a much reduced number of parameters and, consequently, higher precision for estimating expression levels. Thus, we developed a mixed-effects ANOVA model with some random and some fixed effects. It automatically accounts for local normalization between different arrays and for background correction. The method was applied to each of the 6,584 genes investigated in a microarray experiment on two mouse cell lines, PA6/S and PA6/8, where PA6/S enhances proliferation of Pre B cells in vitro but PA6/8 does not. To detect a set of differentially expressed genes (multiple testing problem), we applied the method of controlling the false discovery rate (FDR), which successfully identified 207 genes with significantly different expression levels.

The staufen/pumilio pathway is involved in Drosophila long-term memory.

BACKGROUND: Memory formation after olfactory learning in Drosophila displays behavioral and molecular properties similar to those of other species. Particularly, long-term memory requires CREB-dependent transcription, suggesting the regulation of "downstream" genes. At the cellular level, long-lasting synaptic plasticity in many species also appears to depend on CREB-mediated gene transcription and subsequent structural and functional modification of relevant synapses. To date, little is known about the molecular-genetic mechanisms that contribute to this process during memory formation. RESULTS: We used two complementary strategies to identify these genes. From DNA microarrays, we identified 42 candidate memory genes that appear to be transcriptionally regulated in normal flies during memory formation. Via mutagenesis, we have independently identified 60 mutants with defective long-term memory and have defined molecular lesions for 58 of these. The pumilio translational repressor was found from both approaches, along with six additional genes with established roles in local control of mRNA translation. In vivo disruptions of four genes--staufen, pumilio, oskar, and eIF-5C--yield defective memory. CONCLUSIONS: Convergent findings from our behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification.

Molecular cloning and functional characterization of human vesicular glutamate transporter 3.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.