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.

A prospective, randomized, open label, phase iii clinical trial of novottf-100a vs best standard of care chemotherapy in patients with recurrent glioblastoma

BACKGROUND: Glioblastoma, the most common adult primary malignant brain tumor, confers poor prognosis (median survival of 15 months) notwithstanding aggressive treatment. Combination chemotherapy including carmustine (BCNU) or temozolomide (TMZ) with the MGMT inhibitor O6-benzylguanine (O6BG) has been used, but has been associated with dose-limiting hematopoietic toxicity. OBJECTIVE: To assess safety and efficacy of a retroviral vector encoding the O6BG-resistant MGMTP140K gene for transduction and autologous transplantation of hematopoietic stem cells (HSCs) in MGMT unmethylated, newly diagnosed glioblastoma patients in an attempt to chemoprotect bone marrowduring combination O6BG/TMZ therapy. METHODS: Three patients have been enrolled in the first cohort. Patients underwent standard radiation therapy without TMZ followed by G-CSF mobilization, apheresis, and conditioning with 600 mg/m2 BCNU prior to infusion of gene-modified cells. Posttransplant, patients were treated with 28-day cycles of single doseTMZ (472 mg/m2) with 48-hour intravenous O6BG (120 mg/m2 bolus, then 30 mg/m2/d). RESULTS: The BCNU dose was nonmyeloablative with ANC ,500/mL for ≤3 d and nadir thrombocytopenia of 28,000/mL. Gene marking in pre-infusion colony forming units (CFUs) was 70.6%, 79.0%, and 74.0% in Patients 1, 2, and 3, respectively, by CFU-PCR. Following engraftment, gene marking in white blood cells and sorted granulocytes ranged between 0.37-0.84 and 0.33-0.83 provirus copies, respectively, by real-time PCR. Posttransplant gene marking in CFUs from CD34-selected cells ranged from 28.5% to 47.4%. Patients have received 4, 3, and 2 cycles of O6BG/TMZ, respectively, with evidence for selection of gene-modified cells. One patient has received a single dose-escalated cycle at 590 mg/m2 TMZ. No additional extra-hematopoietic toxicity has been observed thus far and all three patients exhibit stable disease at 7-8 months since diagnosis CONCLUSIONS: We believe that these data demonstrate the feasibility of achieving significant engraftment of MGMTP140K-modified cells with a well-tolerated dose of BCNU. Further follow-up will determine whether this approach will allow for further dose escalation of TMZ and improved survival.

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.

Laboratory estimation of the effects of increasing temperatures on the duration of gonotrophic cycle of Anopheles albimanus (Diptera: Culicidae)

The increase of malaria transmission in the Pacific Coast of Colombia during the occurrence of El Niño warm event has been found not to be linked to increases in the density of the vector Anopheles albimanus, but to other temperature-sensitive variables such as longevity, duration of the gonotrophic cycle or the sporogonic period of Plasmodium. The present study estimated the effects of temperature on duration of the gonotrophic cycle and on maturation of the ovaries of An. albimanus. Blood fed adult mosquitoes were exposed to temperatures of 24, 27, and 30°C, held individually in oviposition cages and assessed at 12 h intervals. At 24, 27, and 30°C the mean development time of the oocytes was 91.2 h (95% C.I.: 86.5-96), 66.2 h (61.5-70.8), and 73.1 h (64-82.3), respectively. The mean duration of the gonotrophic cycle for these three temperatures was 88.4 h (81.88-94.9), 75 h (71.4-78.7), and 69.1 h (64.6-73.6) respectively. These findings indicate that both parameters in An. albimanus are reduced when temperatures rose from 24 to 30°C, in a nonlinear manner. According to these results the increase in malaria transmission during El Niño in Colombia could be associated with a shortening of the gonotrophic cycle in malaria vectors, which could enhance the frequency of man-vector contact, affecting the incidence of the disease.

Framework for Junior Cycle - Full Report

The Framework for Junior Cycle (The Framework) was published in October 2012. It is based on the National Council for Curriculum and Assessment’s advice to the Department as set out in Towards a Framework for Junior Cycle (November 2011) and on research into education for our young people aged 12 to 15 / 16 years. The Framework document can be downloaded from www.education.ie and www.ncca. The mission of the Department of Education and Skills (DES) is “to enable learners to achieve their full potential and contribute to Ireland’s economic, social and cultural development”. By placing students at the centre of the educational experience, the DES wants to ensure that junior cycle education will improve learning experiences and outcomes. The implementation of the Framework for Junior Cycle will enable post-primary schools to provide a quality, inclusive and relevant education with improved learning outcomes for all students, including those with special educational needs

Framework for Junior Cycle 2015

This document, the Framework for Junior Cycle (2015), outlines the key educational changes that the Department of Education and Skills (DES) is putting in place for young people in the first three years of their post-primary education. It builds on and advances the vision for junior cycle reform that was outlined in the Framework for Junior Cycle (2012). The Framework for Junior Cycle (2015) incorporates a shared understanding of how teaching, learning and assessment practices should evolve to support the delivery of a quality, inclusive and relevant education that will meet the needs of junior cycle students, both now and in the future. This shared understanding is informed by engagement with stakeholders and by national and international research.

Framework for the Junior Cycle - Information on the Framework

The Framework for Junior Cycle (The Framework) was published in October 2012. It is based on the National Council for Curriculum and Assessment’s advice to the Department as set out in Towards a Framework for Junior Cycle (November 2011) and on research into education for our young people aged 12 to 15 / 16 years. The Framework document can be downloaded from www.education.ie and www.ncca.

Naming the Reformed Junior Cycle Proposals for the Minister for Education and Skills - Irish Second Level Students Union (ISSU)

It was important to us to engage with as many students as possible throughout the process of developing a new name for the reformed junior cycle. In this vein, we used a wide variety of methods to engage with students in order to capture as many ideas as possible; text messaging, Facebook, Twitter, email and consultation sessions. We circulated posters to all schools via post and/or email, and contacted schools in catchment areas for the consultation sessions by phone. In our consultation sessions, we had discussions with the participating students about what the new junior cycle would be, closely guided by the content of “Towards a Framework for Junior Cycle” from the National Council for Curriculum and assessment. In these sessions, students then gave feedback on what they thought of the reformed junior cycle, developed their own ideas, and identified what they thought should be reflected in the name of the reformed junior cycle

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.

Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

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.

Assessorament als mestres d'Educació Infantil en l'atenció als nens i nenes amb n.e.e. que presenten retards en el desenvolupament o dificultats en els aprenentatges

Aquest projecte pretén demostrar la importància del paper del psicopedagog com a coordinador dels equips d'atenció precoç des d'un enfocament interdisciplinari en què es valora l'àmbit educatiu com una part important de la intervenció precoç o primerenca i en què les interaccions que s'hi generen poden ser una eina per a la prevenció en la primera infància. Per tal de poder demostrar la pertinença d'aquest projecte dins de l'atenció precoç en l'atenció a la mainada a la nostra comarca, he portat a terme una recerca prèvia a les escoles bressol per tal de saber les necessitats reals d'assessorament als mestres d'aquest cicle d'educació Infantil. Així mateix, he fet un seguiment del cas d'un nen de 30 mesos amb tot el que això comporta des d'un tractament interdisciplinari.