Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

<p>Abstract</p> <p>Background</p> <p>Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.</p> <p>The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.</p> <p>Results</p> <p>The expression stability of the candidate reference genes was evaluated using the 2^-ΔC'Tmethod and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.</p

Hábitos y valores del alumnado en centros de primaria de alta eficacia escolar

Diversas investigaciones concluyen que la educación en valores es una parte inseparable de la enseñanza efi caz y que se vincula con mejoras en el aprendizaje y el bienestar emocional y social del alumnado. Este trabajo pretende encontrar evidencias de la relación entre la educación en valores y la efi cacia escolar en escuelas de primaria del País Vasco. Se plantea conocer los hábitos y valores del alumnado de centros eficaces, escuelas que obtienen una puntuación media superior a la esperable una vez controlados, mediante modelos jerárquicos lineales, los efectos de las variables contextuales, e identificar buenas prácticas en el área. Se aplicó un cuestionario a 391 estudiantes (51.9% chicos y 48.1% chicas) y se realizaron entrevistas semiestructuradas a los Equipos Directivos de tres de estos centros. Los resultados confirman que el alumnado tiene una valoración positiva de su nivel de adquisición de hábitos y valores, pero se encontraron diferencias significativas en función del sexo. Además, el aprendizaje cooperativo es la estrategia más utilizada para educar en valores en las aulas. Se concluye el trabajo con las implicaciones para la práctica educativa derivadas tanto de las diferentes percepciones sobre los hábitos y valores, como de una actuación estereotípica de los géneros.Multiple research works conclude that values education is essential for any effective teaching, helps to improve students´ learning and their socioemotional well-being. The objective of this work is to fi nd evidence of the relationship between values education and school effectiveness in elementary schools in the Basque Country. In addition, it will identify the habits and values of elementary school students enrolled in those effective schools with a higher mean score than could be expected once the effects of contextual variables have been monitored by linear hierarchical models, and will pinpoint good practices in the area. A questionnaire was distributed among 391 students (51.9% boys and 48.1% girls) and semistructured interviews were carried out with management teams from three of the schools. The results confi rm that students assess their learning of habits and values positively, but signifi cant differences were found depending on the gender of the participants. Cooperative learning is the strategy most commonly used to teach values in the classrooms. This study offers pedagogical consequences stemming from the different perceptions of habits and values, as well as from a stereotypical performance of the genders

A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke

It remains unclear why some individuals are susceptible to excitotoxicity after stroke. A possible explanation is impaired glutamate uptake. We have found a highly prevalent polymorphism in the promoter of the glutamate transporter EAAT2 gene that abolishes a putative regulatory site for activator protein–2 (AP-2) and creates a new consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2). The mutant genotype is associated with increased plasma glutamate concentrations and with a higher frequency of early neurological worsening in human stroke. After transfection into astrocytes, the mutant promoter was not activated by AP-2 and was effectively repressed by GCF2, and its activity in the presence of GCF2 was reduced when compared with the AP-2–cotransfected wild-type promoter. We also show that GCF2 is expressed in ischemic rat brain, suggesting that decreased glutamate uptake occurs in individuals carrying the mutation after stroke. These findings may explain individual susceptibility to excitotoxic damage after stroke as well as the failure of glutamate antagonists in those patients without this polymorphism

Rosiglitazone-induced CD36 up-regulation resolves inflammation by PPARγ and 5-LO-dependent pathways.

PPARγ-achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5-LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ-induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5-LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5(-/-) mice exposed to distal pMCAO. BMMs were obtained from Lys-M Cre(+) PPARγ(f/f) and Lys-M Cre(-) PPARγ(f/f) mice. Stereological counting of double-immunofluorescence-labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5-LO gene, which is induced in neurons by PPARγ activation and at least by one of its products--LXA4--which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5-LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness CSD2010-00045 (to M.A.M.) SAF2009-08145 and SAF2012-33216 (to M.A.M.), SAF2011-23354 (toI.L.), SAF2009-07466 and SAF2012-31483 (to M.R.), from Fondo Europeo de Desarrollo Regional (FEDER) “Instituto de Salud Carlos III” RETICS RD12/0014/0003 (to I.L. and from the local govern-ment of Madrid S2010/BMD-2336 (to M.A.M.) and S2010/BMD-2349 (to I.L.). I.B. and M.I.C. are fellows of the Spanish Ministry ofEconomy and Competitiveness. The authors thank Tamara Atanesand Roberto Cañadas for their technical assistance.S

First-in-human phase I clinical trial of a TLR4-binding DNA aptamer, ApTOLL: Safety and pharmacokinetics in healthy volunteers.

ApTOLL is an aptamer that antagonizes Toll-like receptor 4 and improves functional outcomes in models of ischemic stroke and myocardial infarction. The aim of this study was to characterize the safety and pharmacokinetics of ApTOLL in healthy volunteers. A first-in-human dose-ascending, randomized, placebo-controlled phase I clinical trial to assess safety and pharmacokinetics of ApTOLL (30-min infusion intravenously) was performed in 46 healthy adult male volunteers. The study was divided into two parts: part A included seven single ascending dose levels, and part B had one multiple dose cohort. Safety and pharmacokinetic parameters were evaluated. No serious adverse events or biochemistry alterations were detected at any dose nor at any administration pattern studied. Maximum concentration was detected at the end of the infusion and mean half-life was 9.3 h. Interestingly, exposure increased in the first four levels receiving doses from 0.7 mg to 14 mg (AUC of 2,441.26 h∗ng/mL to 23,371.11 h∗ng/mL) but remained stable thereafter (mean of 23,184.61 h∗ng/mL after 70 mg). Consequently, the multiple dose study did not show any accumulation of ApTOLL. These results show an excellent safety and adequate pharmacokinetic profile that, together with the efficacy demonstrated in nonclinical studies, provide the basis to start clinical trials in patients.This study was sponsored by aptaTargets S.L. (Madrid, Spain) and was conducted at the Clinical Trials Unit (La Princesa Hospital, Madrid, Spain). The study was supported by a grant from the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6651- 1). Authors acknowledge David Segarra and M. Eugenia Zarabozo (aptaTargets S.L.) for their contribution in the management and funding of the trial, and Alba Singla (Anagram; Barcelona, Spain) for her contribution in the monitoring of the trial.S

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.S

Towards a standardization of biomethane potential tests

8 PáginasProduction of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. A workshop was held in June 2015 in Leysin Switzerland to agree on common solutions to the conundrum of inconsistent BMP test results. A discussion covers actions and criteria that are considered compulsory ito accept and validate a BMP test result; and recommendations concerning the inoculum substrate test setup and data analysis and reporting ito obtain test results that can be validated and reproduced.The workshop in Leysin, Switzerland, has been financed by the Swiss Federal Office for Energy, and co-sponsored by Bioprocess Control Sweden AB, Lund, Sweden. The authors thank Alexandra Maria Murray for editing the English

Vasodilator factors in the systemic and local adaptations to pregnancy

We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, angiotensin-(1–7) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy

Artificial reefs built by 3D printing: Systematisation in the design, material selection and fabrication

The recovery of degraded marine coasts and the improvement of natural habitats are current issues of vital importance for the development of life, both marine and terrestrial. In this sense, the immersion of artificial reefs (ARs) in the marine environment is a way to stimulate the recovery of these damaged ecosystems. But it is necessary to have a multidisciplinary approach that analyses the materials, designs and construction process of artificial reefs in order to understand their true impact on the environment. For this reason, this paper presents the manufacture of artificial reefs by 3D printing, proposing designs with a combination of prismatic and random shapes, with different external overhangs as well as inner holes. For the definition of the artificial reef designs, criteria provided by marine biologists and the results obtained from a numerical simulation with ANSYS were taken into account, with which the stability of the artificial reefs on the seabed was analysed. Three dosages of cement mortars and three dosages of geopolymer mortars were studied as impression materials. The studies included determination of the rheological properties of the mortars, to define the printability, determination of the cost of the materials used, and determination of the mechanical strength and biological receptivity in prismatic specimens that were immersed in the sea for 3 months. To evaluate the environmental impact of the materials used in the production of the mortars, a Life Cycle Assessment (LCA) was carried out. In order to choose the mortars that encompassed the best properties studied, Multi-Criteria Decision Making (MCDM) was applied and the two best mortars were used for the manufacture of the artificial reefs. Finally, the advantages and disadvantages of the 3D printing process used were analysed. The results of the studies carried out in this research show that cement mortars have better characteristics for artificial reef applications using 3D printing, and that the technique applied for the manufacture of the artificial reefs allowed the digital models to be faithfully reproduced