Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Monitor EMG con conexión inalámbrica

El sistema desarrollado en este trabajo permite la detección de fatiga del músculo de un paciente. Las señales electromiográficas generadas por el músculo son detectadas mediante electrodos superficiales, que sirven de entrada al circuito hacia los diferentes canales de entrada. Tras una primera etapa de amplificación, las señales son filtradas por un filtro pasabanda. Las señales acondicionadas se digitalizan mediante un conversor analógico digital que está conectado a un microcontrolador. Éste establece la comunicación con el PC enviándole los datos a través del puerto serie. Finalmente, mediante un programa implementado en Labview se procesan los datos y se determina la existencia o no de fatiga muscular.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models.

BACKGROUND: Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS: We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS: We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Competitive ability not kinship affects growth of Arabidopsis thaliana accessions.

In many organisms, individuals behave more altruistically towards relatives than towards unrelated individuals. Here, we conducted a study to determine if the performance of Arabidopsis thaliana is influenced by whether individuals are in competition with kin or non-kin. We selected seven pairs of genetically distinct accessions that originated from local populations throughout Europe. We measured the biomass of one focal plant surrounded by six kin or non-kin neighbours in in vitro growth experiments and counted the number of siliques produced per pot by one focal plant surrounded by four kin or non-kin neighbours. The biomass and number of siliques of a focal plant were not affected by the relatedness of the neighbour. Depending on the accession, a plant performed better or worse in a pure stand than when surrounded by non-kin plants. In addition, whole-genome microarray analyses revealed that there were no genes differentially expressed between kin and non-kin conditions. In conclusion, our study does not provide any evidence for a differential response to kin vs non-kin in A. thaliana. Rather, the outcome of the interaction between kin and non-kin seems to depend on the strength of the competitive abilities of the accessions.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Effect of training-sample size and classification difficulty on the accuracy of genomic predictors.

Introduction: As part of the MicroArray Quality Control (MAQC)-II project, this analysis examines how the choice of univariate feature-selection methods and classification algorithms may influence the performance of genomic predictors under varying degrees of prediction difficulty represented by three clinically relevant endpoints. Methods: We used gene-expression data from 230 breast cancers (grouped into training and independent validation sets), and we examined 40 predictors (five univariate feature-selection methods combined with eight different classifiers) for each of the three endpoints. Their classification performance was estimated on the training set by using two different resampling methods and compared with the accuracy observed in the independent validation set. Results: A ranking of the three classification problems was obtained, and the performance of 120 models was estimated and assessed on an independent validation set. The bootstrapping estimates were closer to the validation performance than were the cross-validation estimates. The required sample size for each endpoint was estimated, and both gene-level and pathway-level analyses were performed on the obtained models. Conclusions: We showed that genomic predictor accuracy is determined largely by an interplay between sample size and classification difficulty. Variations on univariate feature-selection methods and choice of classification algorithm have only a modest impact on predictor performance, and several statistically equally good predictors can be developed for any given classification problem.

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.

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

Electroencephalographic source imaging: a prospective study of 152 operated epileptic patients.

Electroencephalography is mandatory to determine the epilepsy syndrome. However, for the precise localization of the irritative zone in patients with focal epilepsy, costly and sometimes cumbersome imaging techniques are used. Recent small studies using electric source imaging suggest that electroencephalography itself could be used to localize the focus. However, a large prospective validation study is missing. This study presents a cohort of 152 operated patients where electric source imaging was applied as part of the pre-surgical work-up allowing a comparison with the results from other methods. Patients (n = 152) with >1 year postoperative follow-up were studied prospectively. The sensitivity and specificity of each imaging method was defined by comparing the localization of the source maximum with the resected zone and surgical outcome. Electric source imaging had a sensitivity of 84% and a specificity of 88% if the electroencephalogram was recorded with a large number of electrodes (128-256 channels) and the individual magnetic resonance image was used as head model. These values compared favourably with those of structural magnetic resonance imaging (76% sensitivity, 53% specificity), positron emission tomography (69% sensitivity, 44% specificity) and ictal/interictal single-photon emission-computed tomography (58% sensitivity, 47% specificity). The sensitivity and specificity of electric source imaging decreased to 57% and 59%, respectively, with low number of electrodes (<32 channels) and a template head model. This study demonstrated the validity and clinical utility of electric source imaging in a large prospective study. Given the low cost and high flexibility of electroencephalographic systems even with high channel counts, we conclude that electric source imaging is a highly valuable tool in pre-surgical epilepsy evaluation.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency.

Microsatellite instability (MSI) occurs in 10-20% of colorectal tumours and is associated with good prognosis. Here we describe the development and validation of a genomic signature that identifies colorectal cancer patients with MSI caused by DNA mismatch repair deficiency with high accuracy. Microsatellite status for 276 stage II and III colorectal tumours has been determined. Full-genome expression data was used to identify genes that correlate with MSI status. A subset of these samples (n = 73) had sequencing data for 615 genes available. An MSI gene signature of 64 genes was developed and validated in two independent validation sets: the first consisting of frozen samples from 132 stage II patients; and the second consisting of FFPE samples from the PETACC-3 trial (n = 625). The 64-gene MSI signature identified MSI patients in the first validation set with a sensitivity of 90.3% and an overall accuracy of 84.8%, with an AUC of 0.942 (95% CI, 0.888-0.975). In the second validation, the signature also showed excellent performance, with a sensitivity 94.3% and an overall accuracy of 90.6%, with an AUC of 0.965 (95% CI, 0.943-0.988). Besides correct identification of MSI patients, the gene signature identified a group of MSI-like patients that were MSS by standard assessment but MSI by signature assessment. The MSI-signature could be linked to a deficient MMR phenotype, as both MSI and MSI-like patients showed a high mutation frequency (8.2% and 6.4% of 615 genes assayed, respectively) as compared to patients classified as MSS (1.6% mutation frequency). The MSI signature showed prognostic power in stage II patients (n = 215) with a hazard ratio of 0.252 (p = 0.0145). Patients with an MSI-like phenotype had also an improved survival when compared to MSS patients. The MSI signature was translated to a diagnostic microarray and technically and clinically validated in FFPE and frozen samples.