Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain : validation and literature search

La douleur neuropathique est définie comme une douleur causée par une lésion du système nerveux somato-sensoriel. Elle se caractérise par des douleurs exagérées, spontanées, ou déclenchées par des stimuli normalement non douloureux (allodynie) ou douloureux (hyperalgésie). Bien qu'elle concerne 7% de la population, ses mécanismes biologiques ne sont pas encore élucidés. L'étude des variations d'expressions géniques dans les tissus-clés des voies sensorielles (notamment le ganglion spinal et la corne dorsale de la moelle épinière) à différents moments après une lésion nerveuse périphérique permettrait de mettre en évidence de nouvelles cibles thérapeutiques. Elles se détectent de manière sensible par reverse transcription quantitative real-time polymerase chain reaction (RT- qPCR). Pour garantir des résultats fiables, des guidelines ont récemment recommandé la validation des gènes de référence utilisés pour la normalisation des données ("Minimum information for publication of quantitative real-time PCR experiments", Bustin et al 2009). Après recherche dans la littérature des gènes de référence fréquemment utilisés dans notre modèle de douleur neuropathique périphérique SNI (spared nerve injury) et dans le tissu nerveux en général, nous avons établi une liste de potentiels bons candidats: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) et L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) et hydroxymethyl-bilane synthase (HMBS). Nous avons évalué la stabilité d'expression de ces gènes dans le ganglion spinal et dans la corne dorsale à différents moments après la lésion nerveuse (SNI) en calculant des coefficients de variation et utilisant l'algorithme geNorm qui compare les niveaux d'expression entre les différents candidats et détermine la paire de gènes restante la plus stable. Il a aussi été possible de classer les gènes selon leur stabilité et d'identifier le nombre de gènes nécessaires pour une normalisation la plus précise. Les gènes les plus cités comme référence dans le modèle SNI ont été GAPDH, HMBS, Actb, HPRT1 et 18S. Seuls HPRT1 and 18S ont été précédemment validés dans des arrays de RT-qPCR. Dans notre étude, tous les gènes testés dans le ganglion spinal et dans la corne dorsale satisfont au critère de stabilité exprimé par une M-value inférieure à 1. Par contre avec un coefficient de variation (CV) supérieur à 50% dans le ganglion spinal, 18S ne peut être retenu. La paire de gènes la plus stable dans le ganglion spinal est HPRT1 et Actb et dans la corne dorsale il s'agit de RPL29 et RPL13a. L'utilisation de 2 gènes de référence stables suffit pour une normalisation fiable. Nous avons donc classé et validé Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 et 18S comme gènes de référence utilisables dans la corne dorsale pour le modèle SNI chez le rat. Dans le ganglion spinal 18S n'a pas rempli nos critères. Nous avons aussi déterminé que la combinaison de deux gènes de référence stables suffit pour une normalisation précise. Les variations d'expression génique de potentiels gènes d'intérêts dans des conditions expérimentales identiques (SNI, tissu et timepoints post SNI) vont pouvoir se mesurer sur la base d'une normalisation fiable. Non seulement il sera possible d'identifier des régulations potentiellement importantes dans la genèse de la douleur neuropathique mais aussi d'observer les différents phénotypes évoluant au cours du temps après lésion nerveuse.

Real-time antifungal susceptibility testing of Mucor spp., Fusarium spp. and Scedosporium spp. by isothermal microcalorimetry

Objective: Aspergillus species are the main pathogens causing invasive fungal infections but the prevalence of other mould species is rising. Resistance to antifungals among these new emerging pathogens presents a challenge for managing of infections. Conventional susceptibility testing of non-Aspergillus species is laborious and often difficult to interpret. We evaluated a new method for real-time susceptibility testing of moulds based on their of growth-related heat production.Methods: Laboratory and clinical strains of Mucor spp. (n = 4), Scedoporium spp. (n = 4) and Fusarium spp. (n = 5) were used. Conventional MIC was determined by microbroth dilution. Isothermal microcalorimetry was performed at 37 C using Sabouraud dextrose broth (SDB) inoculated with 104 spores/ml (determined by microscopical enumeration). SDB without antifungals was used for evaluation of growth characteristics. Detection time was defined as heat flow exceeding 10 lW. For susceptibility testing serial dilutions of amphotericin B, voriconazole, posaconazole and caspofungin were used. The minimal heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration, inhbiting 50% of the heat produced by the growth control at 48 h or at 24 h for Mucor spp. Susceptibility tests were performed in duplicate.Results: Tested mould genera had distinctive heat flow profiles with a median detection time (range) of 3.4 h (1.9-4.1 h) for Mucor spp, 11.0 h (7.1-13.7 h) for Fusarium spp and 29.3 h (27.4-33.0 h) for Scedosporium spp. Graph shows heat flow (in duplicate) of one representative strain from each genus (dashed line marks detection limit). Species belonging to the same genus showed similar heat production profiles. Table shows MHIC and MIC ranges for tested moulds and antifungals.Conclusions: Microcalorimetry allowed rapid detection of growth of slow-growing species, such as Fusarium spp. and Scedosporium spp. Moreover, microcalorimetry offers a new approach for antifungal susceptibility testing of moulds, correlating with conventional MIC values. Interpretation of calorimetric susceptibility data is easy and real-time data on the effect of different antifungals on the growth of the moulds is additionally obtained. This method may be used for investigation of different mechanisms of action of antifungals, new substances and drug-drug combinations.

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay.

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology. A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory. A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10PFU/ml (Arm53b and WE54) and 1PFU/ml (Traub and E350). Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses. This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R²) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Estudi del disseny i la implementació d¿un: Sistema de control de la salut dels bancs europeus en temps real.

El Banc Central Europeu(BCE) ens ha encarregat preparar-li una base de dades on poder emmagatzemar uns registres diaris relacionats amb uns indicadors. En el moment d¿enregistrar els indicadors si es viola alguna regla de negoci, prèviament definides per el BCE, es llencen una sèrie d¿alertes que també s¿emmagatzemaran a la base de dades per al seu posterior tractament. Amb la informació emmagatzemada en aquesta base de dades(BD), el BCE utilitzant una aplicació per explotar aquestes dades, podrà controlar la salut dels bancs europeus en temps real i facilitar la presa de decisions. També se¿ns demana la implementació d¿un mòdul estadístic on hi hagin una sèrie de dades ja precalculades per facilitar la seva consulta.

Virtual library, a real library?

Les possibilitats ofertes per la virtualitat tenen una gran importància en l'esfera educativa i en tots els aspectes referents a ella. Evidentment, les biblioteques i els centres de documentació no són estranys a aquest nou ambient virtual facilitat pel canvi social, econòmic i, sobretot, tecnològic que ha permès que els bibliotecaris-documentalistes accedeixin a gran quantitat d'informació i de documentació, permetent que actuïn com a agents intermediaris entre aquesta nova situació i l'ús que se'n poden fer pels diversos tipus d'usuaris.

Semi-online neural-Q_leaming for real-time robot learning

Reinforcement learning (RL) is a very suitable technique for robot learning, as it can learn in unknown environments and in real-time computation. The main difficulties in adapting classic RL algorithms to robotic systems are the generalization problem and the correct observation of the Markovian state. This paper attempts to solve the generalization problem by proposing the semi-online neural-Q_learning algorithm (SONQL). The algorithm uses the classic Q_learning technique with two modifications. First, a neural network (NN) approximates the Q_function allowing the use of continuous states and actions. Second, a database of the most representative learning samples accelerates and stabilizes the convergence. The term semi-online is referred to the fact that the algorithm uses the current but also past learning samples. However, the algorithm is able to learn in real-time while the robot is interacting with the environment. The paper shows simulated results with the "mountain-car" benchmark and, also, real results with an underwater robot in a target following behavior

Policy gradient based Reinforcement Learning for real autonomous underwater cable tracking

This paper proposes a field application of a high-level reinforcement learning (RL) control system for solving the action selection problem of an autonomous robot in cable tracking task. The learning system is characterized by using a direct policy search method for learning the internal state/action mapping. Policy only algorithms may suffer from long convergence times when dealing with real robotics. In order to speed up the process, the learning phase has been carried out in a simulated environment and, in a second step, the policy has been transferred and tested successfully on a real robot. Future steps plan to continue the learning process on-line while on the real robot while performing the mentioned task. We demonstrate its feasibility with real experiments on the underwater robot ICTINEU AUV

Towards a real-time vision-based navigation system for a small-class UUV

This paper deals with the problem of navigation for an unmanned underwater vehicle (UUV) through image mosaicking. It represents a first step towards a real-time vision-based navigation system for a small-class low-cost UUV. We propose a navigation system composed by: (i) an image mosaicking module which provides velocity estimates; and (ii) an extended Kalman filter based on the hydrodynamic equation of motion, previously identified for this particular UUV. The obtained system is able to estimate the position and velocity of the robot. Moreover, it is able to deal with visual occlusions that usually appear when the sea bottom does not have enough visual features to solve the correspondence problem in a certain area of the trajectory

A new approach to real time traffic sign recognition based on colour information

The automatic interpretation of conventional traffic signs is very complex and time consuming. The paper concerns an automatic warning system for driving assistance. It does not interpret the standard traffic signs on the roadside; the proposal is to incorporate into the existing signs another type of traffic sign whose information will be more easily interpreted by a processor. The type of information to be added is profuse and therefore the most important object is the robustness of the system. The basic proposal of this new philosophy is that the co-pilot system for automatic warning and driving assistance can interpret with greater ease the information contained in the new sign, whilst the human driver only has to interpret the "classic" sign. One of the codings that has been tested with good results and which seems to us easy to implement is that which has a rectangular shape and 4 vertical bars of different colours. The size of these signs is equivalent to the size of the conventional signs (approximately 0.4 m2). The colour information from the sign can be easily interpreted by the proposed processor and the interpretation is much easier and quicker than the information shown by the pictographs of the classic signs

The Estrada Real project and endemic diseases: the case of schistosomiasis, geoprocessing and tourism

Geographical Information System (GIS) is a tool that has recently been applied to better understand spatial disease distributions. Using meteorological, social, sanitation, mollusc distribution data and remote sensing variables, this study aimed to further develop the GIS technology by creating a model for the spatial distribution of schistosomiasis and to apply this model to an area with rural tourism in the Brazilian state of Minas Gerais (MG). The Estrada Real, covering about 1,400 km, is the largest and most important Brazilian tourism project, involving 163 cities in MG with different schistosomiasis prevalence rates. The model with three variables showed a R² = 0.34, with a standard deviation of risk estimated adequate for public health needs. The main variables selected for modelling were summer vegetation, summer minimal temperature and winter minimal temperature. The results confirmed the importance of Remote Sensing data and the valuable contribution of GIS in identifying priority areas for intervention in tourism regions which are endemic to schistosomiasis.

Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

This study aimed to quantify Toxoplasma gondii in tissue samples of serologically positive chickens using real-time polymerase chain reaction (PCR). Of 65 chickens evaluated, 28 were positive for T. gondii antibodies. Brain and heart samples were collected from 26 seropositive chickens and DNA was extracted using Trizol® and amplified using real-time PCR with SYBR® Green. Parasite DNA was detected in 24 of the 26 samples analyzed; the number of positive tissue samples and the parasite quantity did not differ between tissue types. The results confirmed the analytical sensitivity of parasite detection in chicken tissue samples and demonstrated the possibility of using other molecular systems for genotypic analysis.

Using 3D surface datasets to understand landslide evolution: From analogue models to real case study

Early detection of landslide surface deformation with 3D remote sensing techniques, as TLS, has become a great challenge during last decade. To improve our understanding of landslide deformation, a series of analogue simulation have been carried out on non-rigid bodies coupled with 3D digitizer. All these experiments have been carried out under controlled conditions, as water level and slope angle inclination. We were able to follow 3D surface deformation suffered by complex landslide bodies from precursory deformation still larger failures. These experiments were the basis for the development of a new algorithm for the quantification of surface deformation using automatic tracking method on discrete points of the slope surface. To validate the algorithm, comparisons were made between manually obtained results and algorithm surface displacement results. Outputs will help in understanding 3D deformation during pre-failure stages and failure mechanisms, which are fundamental aspects for future implementation of 3D remote sensing techniques in early warning systems.

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.