Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

Substantial deletion overlap among divergent Arabidopsis genomes revealed by intersection of short reads and tiling arrays.

ABSTRACT: Identification of small polymorphisms from next generation sequencing short read data is relatively easy, but detection of larger deletions is less straightforward. Here, we analyzed four divergent Arabidopsis accessions and found that intersection of absent short read coverage with weak tiling array hybridization signal reliably flags deletions. Interestingly, individual deletions were frequently observed in two or more of the accessions examined, suggesting that variation in gene content partly reflects a common history of deletion events.

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays.

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl(-1) of RNA material, without prior PCR amplification and use of labels.

Genome-wide arrays in routine diagnostics of hematological malignancies.

Over the last three decades, cytogenetic analysis of malignancies has become an integral part of disease evaluation and prediction of prognosis or responsiveness to therapy. In most diagnostic laboratories, conventional karyotyping, in conjunction with targeted fluorescence in situ hybridization analysis, is routinely performed to detect recurrent aberrations with prognostic implications. However, the genetic complexity of cancer cells requires a sensitive genome-wide analysis, enabling the detection of small genomic changes in a mixed cell population, as well as of regions of homozygosity. The advent of comprehensive high-resolution genomic tools, such as molecular karyotyping using comparative genomic hybridization or single-nucleotide polymorphism microarrays, has overcome many of the limitations of traditional cytogenetic techniques and has been used to study complex genomic lesions in, for example, leukemia. The clinical impact of the genomic copy-number and copy-neutral alterations identified by microarray technologies is growing rapidly and genome-wide array analysis is evolving into a diagnostic tool, to better identify high-risk patients and predict patients' outcomes from their genomic profiles. Here, we review the added clinical value of an array-based genome-wide screen in leukemia, and discuss the technical challenges and an interpretation workflow in applying arrays in the acquired cytogenetic diagnostic setting.

Mixed mating in androdioecious Mercurialis annua inferred using progeny arrays and diploid-acting microsatellite loci in a hexaploid background.

Background and Aims The frequency at which males can be maintained with hermaphrodites in androdioecious populations is predicted to depend on the selfing rate, because self-fertilization by hermaphrodites reduces prospective siring opportunities for males. In particular, high selfing rates by hermaphrodites are expected to exclude males from a population. Here, the first estimates are provided of the mating system from two wild hexaploid populations of the androdioecious European wind-pollinated plant M. annua with contrasting male frequencies.Methods Four diploid microsatellite loci were used to genotype 19-20 progeny arrays from two populations of M. annua, one with males and one without. Mating-system parameters were estimated using the program MLTR.Key Results Both populations had similar, intermediate outcrossing rates (t(m) = 0.64 and 0.52 for the population with and without males, respectively). The population without males showed a lower level of correlated paternity and biparental inbreeding and higher allelic richness and gene diversity than the population with males.Conclusions The results demonstrate the utility of new diploid microsatellite loci for mating system analysis in a hexaploid plant. It would appear that androdioecious M. annua has a mixed-mating system in the wild, an uncommon finding for wind-pollinated species. This study sets a foundation for future research to assess the relative importance of the sexual system, plant-density variation and stochastic processes for the regulation of male frequencies in M. annua over space and time.

Identification and validation of copy number variants using SNP genotyping arrays from a large clinical cohort.

BACKGROUND: Genotypes obtained with commercial SNP arrays have been extensively used in many large case-control or population-based cohorts for SNP-based genome-wide association studies for a multitude of traits. Yet, these genotypes capture only a small fraction of the variance of the studied traits. Genomic structural variants (GSV) such as Copy Number Variation (CNV) may account for part of the missing heritability, but their comprehensive detection requires either next-generation arrays or sequencing. Sophisticated algorithms that infer CNVs by combining the intensities from SNP-probes for the two alleles can already be used to extract a partial view of such GSV from existing data sets. RESULTS: Here we present several advances to facilitate the latter approach. First, we introduce a novel CNV detection method based on a Gaussian Mixture Model. Second, we propose a new algorithm, PCA merge, for combining copy-number profiles from many individuals into consensus regions. We applied both our new methods as well as existing ones to data from 5612 individuals from the CoLaus study who were genotyped on Affymetrix 500K arrays. We developed a number of procedures in order to evaluate the performance of the different methods. This includes comparison with previously published CNVs as well as using a replication sample of 239 individuals, genotyped with Illumina 550K arrays. We also established a new evaluation procedure that employs the fact that related individuals are expected to share their CNVs more frequently than randomly selected individuals. The ability to detect both rare and common CNVs provides a valuable resource that will facilitate association studies exploring potential phenotypic associations with CNVs. CONCLUSION: Our new methodologies for CNV detection and their evaluation will help in extracting additional information from the large amount of SNP-genotyping data on various cohorts and use this to explore structural variants and their impact on complex traits.

Example of Data Telemetry for Biomedical Applications: An In Vivo Experiment

This letter describes a data telemetry biomedical experiment. An implant, consisting of a biometric data sensor, electronics, an antenna, and a biocompatible capsule, is described. All the elements were co-designed in order to maximize the transmission distance. The device was implanted in a pig for an in vivo experiment of temperature monitoring.

Structured RNAs in the ENCODE selected regions of the human genome.

Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack characteristic signals in primary sequence, comparative approaches evaluating evolutionary conservation of structures are most promising. We have used three recently introduced programs based on either phylogenetic-stochastic context-free grammar (EvoFold) or energy directed folding (RNAz and AlifoldZ), yielding several thousand candidate structures (corresponding to approximately 2.7% of the ENCODE regions). EvoFold has its highest sensitivity in highly conserved and relatively AU-rich regions, while RNAz favors slightly GC-rich regions, resulting in a relatively small overlap between methods. Comparison with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3'-UTRs. While we estimate a significant false discovery rate of approximately 50%-70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz and EvoFold, and an additional 239 RNAz or EvoFold predictions are supported by the (more stringent) AlifoldZ algorithm. Five hundred seventy RNAz structure predictions fall into regions that show signs of selection pressure also on the sequence level (i.e., conserved elements). More than 700 predictions overlap with noncoding transcripts detected by oligonucleotide tiling arrays. One hundred seventy-five selected candidates were tested by RT-PCR in six tissues, and expression could be verified in 43 cases (24.6%).

3D geoelectrical monitoring to follow hydric soil behaviour during rainfall

RESUME Durant les dernières années, les méthodes électriques ont souvent été utilisées pour l'investigation des structures de subsurface. L'imagerie électrique (Electrical Resistivity Tomography, ERT) est une technique de prospection non-invasive et spatialement intégrée. La méthode ERT a subi des améliorations significatives avec le développement de nouveaux algorithmes d'inversion et le perfectionnement des techniques d'acquisition. La technologie multicanale et les ordinateurs de dernière génération permettent la collecte et le traitement de données en quelques heures. Les domaines d'application sont nombreux et divers: géologie et hydrogéologie, génie civil et géotechnique, archéologie et études environnementales. En particulier, les méthodes électriques sont souvent employées dans l'étude hydrologique de la zone vadose. Le but de ce travail est le développement d'un système de monitorage 3D automatique, non- invasif, fiable, peu coûteux, basé sur une technique multicanale et approprié pour suivre les variations de résistivité électrique dans le sous-sol lors d'événements pluvieux. En raison des limitations techniques et afin d'éviter toute perturbation physique dans la subsurface, ce dispositif de mesure emploie une installation non-conventionnelle, où toutes les électrodes de courant sont placées au bord de la zone d'étude. Le dispositif le plus approprié pour suivre les variations verticales et latérales de la résistivité électrique à partir d'une installation permanente a été choisi à l'aide de modélisations numériques. Les résultats démontrent que le dispositif pôle-dipôle offre une meilleure résolution que le dispositif pôle-pôle et plus apte à détecter les variations latérales et verticales de la résistivité électrique, et cela malgré la configuration non-conventionnelle des électrodes. Pour tester l'efficacité du système proposé, des données de terrain ont été collectées sur un site d'étude expérimental. La technique de monitorage utilisée permet de suivre le processus d'infiltration 3D pendant des événements pluvieux. Une bonne corrélation est observée entre les résultats de modélisation numérique et les données de terrain, confirmant par ailleurs que le dispositif pôle-dipôle offre une meilleure résolution que le dispositif pôle-pôle. La nouvelle technique de monitorage 3D de résistivité électrique permet de caractériser les zones d'écoulement préférentiel et de caractériser le rôle de la lithologie et de la pédologie de manière quantitative dans les processus hydrologiques responsables d'écoulement de crue. ABSTRACT During the last years, electrical methods were often used for the investigation of subsurface structures. Electrical resistivity tomography (ERT) has been reported to be a useful non-invasive and spatially integrative prospecting technique. The ERT method provides significant improvements, with the developments of new inversion algorithms, and the increasing efficiency of data collection techniques. Multichannel technology and powerful computers allow collecting and processing resistivity data within few hours. Application domains are numerous and varied: geology and hydrogeology, civil engineering and geotechnics, archaeology and environmental studies. In particular, electrical methods are commonly used in hydrological studies of the vadose zone. The aim of this study was to develop a multichannel, automatic, non-invasive, reliable and inexpensive 3D monitoring system designed to follow electrical resistivity variations in soil during rainfall. Because of technical limitations and in order to not disturb the subsurface, the proposed measurement device uses a non-conventional electrode set-up, where all the current electrodes are located near the edges of the survey grid. Using numerical modelling, the most appropriate arrays were selected to detect vertical and lateral variations of the electrical resistivity in the framework of a permanent surveying installation system. The results show that a pole-dipole array has a better resolution than a pole-pole array and can successfully follow vertical and lateral resistivity variations despite the non-conventional electrode configuration used. Field data are then collected at a test site to assess the efficiency of the proposed monitoring technique. The system allows following the 3D infiltration processes during a rainfall event. A good correlation between the results of numerical modelling and field data results can be observed since the field pole-dipole data give a better resolution image than the pole-pole data. The new device and technique makes it possible to better characterize the zones of preferential flow and to quantify the role of lithology and pedology in flood- generating hydrological processes.

IQRray, a new method for Affymetrix microarray quality control, and the homologous organ conservation score, a new benchmark method for quality control metrics.

MOTIVATION: Microarray results accumulated in public repositories are widely reused in meta-analytical studies and secondary databases. The quality of the data obtained with this technology varies from experiment to experiment, and an efficient method for quality assessment is necessary to ensure their reliability. RESULTS: The lack of a good benchmark has hampered evaluation of existing methods for quality control. In this study, we propose a new independent quality metric that is based on evolutionary conservation of expression profiles. We show, using 11 large organ-specific datasets, that IQRray, a new quality metrics developed by us, exhibits the highest correlation with this reference metric, among 14 metrics tested. IQRray outperforms other methods in identification of poor quality arrays in datasets composed of arrays from many independent experiments. In contrast, the performance of methods designed for detecting outliers in a single experiment like Normalized Unscaled Standard Error and Relative Log Expression was low because of the inability of these methods to detect datasets containing only low-quality arrays and because the scores cannot be directly compared between experiments. AVAILABILITY AND IMPLEMENTATION: The R implementation of IQRray is available at: ftp://lausanne.isb-sib.ch/pub/databases/Bgee/general/IQRray.R. CONTACT: Marta.Rosikiewicz@unil.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sequence conservation in Plasmodium falciparum alpha-helical coiled coil domains proposed for vaccine development.

BACKGROUND: The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to alpha-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative alpha-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB. METHODOLOGY/PRINCIPAL FINDINGS: 14 alpha-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 alpha-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative alpha-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for alpha-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved alpha-helical coiled coil motifs. CONCLUSION/SIGNIFICANCE: We conclude that the selection of alpha-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.