Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts

Quantification of the Electrophilicities of Diazoalkanes: Kinetics and Mechanism of Azo Couplings with Enamines and Sulfonium Ylides

Kinetics and mechanism of the reactions of methyl diazoacetate, dimethyl diazomalonate, 4-nitrophenyldiazomethane, and diphenyldiazomethane with sulfonium ylides and enamines were investigated by UV-Vis and NMR spectroscopy. Ordinary alkenes undergo 1,3-dipolar cycloadditions with these diazo compounds. In contrast, sulfonium ylides and enamines attack at the terminal nitrogen of the diazo alkanes to give zwitterions, which undergo various subsequent reactions. As only one new bond is formed in the rate-determining step of these reactions, the correlation lg k(2)(20 degrees C)=s(N)(N+E) could be used to determine the one-bond electrophilicities E of the diazo compounds from the measured second-order rate constants and the known reactivity indices N and s(N) of the sulfonium ylides and enamines. The resulting electrophilicity parameters (-21<E<-18), which are 11-14 orders of magnitude smaller than that of the benzenediazonium ion, are used to define the scope of one-bond nucleophiles which may react with these diazoalkanes

An Overlooked Pathway in 1,3-Dipolar Cycloadditions of Diazoalkanes with Enamines

Methyl diazoacetate reacts with 1-(N-pyrrolidino)cycloalkenes to give products of 1,3-dipolar cycloadditions and azo couplings. The kinetics and mechanisms of these reactions were investigated by NMR spectroscopy and DFT calculations. Orthogonal pi-systems in the 1,3-dipoles of the propargyl-allenyl type allow for two separate reaction pathways for the (3+2)-cycloadditions. The commonly considered concerted pathway is rationalized by the interaction of the enamine HOMO with LUMO+1, the lowest unoccupied orbital of the heteropropargyl anion fragment of methyl diazoacetate. We show that HOMO/LUMO(pi*(N=N)) interactions between enamines and methyl diazoacetate open a previously unrecognized reaction path for stepwise cycloadditions through zwitterionic intermediates with barriers approximately 40 kJ mol(-1) lower in energy in CHCl3 (DFT calculations) than for the concerted path

How well can a seasonal forecast system represent 3 hourly compound wind and precipitation extremes over Europe?

This is the author accepted manuscript. The final version is available on open access from IOP Publishing via the DOI in this recordExtreme precipitation and winds can have a severe impact on society, particularly when they occur at the same place and time. In this study the Met Office's Global Seasonal forecast system version 5 (GloSea5) model ensembles are evaluated against the reanalysis dataset ERA5, to find out how well they represent 3 hourly extreme precipitation, extreme wind and extreme co-occurring events over Europe. Although substantial differences in magnitude are found between precipitation and wind extremes between the datasets, the conditional probability of exceedance above the 99th percentile, which measures the co-occurrence between the two extremes, compares well spatially over Europe. However, significant differences in frequency are found around and over some areas of high topography. Generally GloSea5 underestimates this co-occurrence over sea. The model's co-occurring events at individual locations investigated occur with very similar synoptic patterns to ERA5, indicating that the compound extremes are produced for the correct reasons.University of Exeter College of Engineering, Mathematics and Physical SciencesMet Office Hadley Centre Climate Programm

Compound precipitation and wind extremes over Europe and their relationship to extratropical cyclones

This is the final version. Available on open access from Elsevier via the DOI in this recordWe acknowledge the data providers in the ECA&D project. Klein Tank, A.M.G. and Coauthors, 2002. Daily dataset of 20th-century surface air temperature and precipitation series for the European Climate Assessment. Int. J. of Climatol., 22, 1441–1453. Data and metadata available at https://www.ecad.euWe acknowledge the E-OBS dataset and the data providers in the ECA&D project (https://www.ecad.eu). Cornes, R., G. van der Schrier, E.J.M. van den Besselaar, and P.D. Jones. 2018: An Ensemble Version of the E-OBS Temperature and Precipitation Datasets, J. Geophys. Res. Atmos., 123. doi:10.1029/2017JD028200Extratropical cyclones and their associated extreme precipitation and winds can have a severe impact on society and the co-occurrence between the two extremes is important when assessing risk. In this study the extremal dependency measure, χ, is used to quantify the co-occurrence of extreme precipitation and wind gusts, and is investigated at individual grid points and spatially over Europe. Results using three observational datasets and a higher spatial and temporal resolution version of ERA5 than previously used confirm previous studies. Over Europe high co-occurrence is found over western coasts and low co-occurrence is found over eastern coasts. All datasets have qualitatively similar spatial patterns over most regions of Europe excluding some regions of high topography where ERA5 χ values are much larger. ERA5 represents the timings of daily extreme co-occurring events well, compared to observations. The differences in precipitation accumulation timescales are also accounted for by considering hourly, 6, 24 and 48 hourly co-occurrence. In a few regions co-occurrence changes with longer accumulations, indicating the different speeds and sizes of weather systems affecting these regions. χ in most regions has little increase by allowing a 24 h lag and lead between the precipitation and wind, with a few exceptions where χ is increased by up to 24%. Regions with the larger of these increases are on or around elevated topography. Using an objective feature tracking method, insight into the spatial pattern of extreme precipitation and wind within cyclones over Europe is given. As well as suggesting how many hours apart the extremes occur from one another in a particular location. Extreme co-occurring events are associated with cyclones far more of the time than non extreme events. Given an extreme co-occurring event the chance of a cyclone being within 1110 km is more than 70% for much of Europe. Regions with low co-occurrence have extremes caused by different weather systems and regions with large co-occurrence have both extremes caused by the same weather system. Cyclones linked to extreme events, particularly co-occurring and extreme wind, have larger intensity than those not and for most of Europe these cyclones also have faster mean speed.Natural Environment Research Council (NERC)Met Office Hadley Centre Climate Programm

Making school happen: children-parent-teacher

The exercise of citizenship is today understood as a duty and as a right to be enjoyed within any educational context. Within the school, all of its protagonists are invited to exercise practices of citizenship. No one is excluded; even the less important parties have the right to participate in decisions that, for some reason, may have an influence on their academic life. The citizenship of the child is, thus, a challenge to the changing political, social and educational structures, to the transformation of institutions and to cultural renewal. The existence of harmonious relations between the educational community, the school, the children and the family is dependent on everyone’s ability to understand and communicate with each other. Parents and teachers have made a commitment to a fruitful and unison dialogue on behalf of the quality of education. In this article, we set out from an analysis of the new social realities and of the different meanings assigned to education, to afterwards reflect upon the current educational values and upon the practices that are consistent with those purposes. Citizenship, as well as autonomy, rise, thus, as central concepts, in which each educational community finds reasons for Making School Happen.CIEC – Research Centre on Child Studies, UM (FCT R&D 317

Quantification du contrôle des séquences par la tectonique et l’eustatisme dans le bassin du Dniepr-Donets et sur la plate-forme russe pendant le Carbonifère et le Permien

Introduction. – Une analyse quantitative comparative de la subsidence dans les bassins d’âge paléozoïque supérieur de Moscou (MB) et du Dniepr-Donets (DDB) apporte une vision nouvelle sur l’importance relative de la tectonique et de l’eustatisme comme contrôle de la sédimentation et du fonctionnement de ces bassins. Les résultats publiés sur le segment du Dniepr [Stovba et al., 1995 ; van Wees et al., 1996] sont comparés à de nouveaux résultats provenant du MB et de la partie orientale du DDB (segments du Donets et du Donbass) en utilisant le programme AQUASUB du BRGM. Le bassin de Moscou (MB). – Le MB est situé dans la partie occidentale de la plate-forme russe (fig. 1). Le Carbonifère (fig. 2) y est représenté par environ 650 m de sédiments principalement carbonatés d’origine marine. Une lacune stratigraphique et une érosion importante y sont connues entre le Serpukhovien et le Bashkirien supérieur. La figure 2 présente les séquences du second ordre du MB [Briand et al., 1998] et leur corrélation avec les séquences glaciaires et interglaciaires du Gondwana [Lopez-Gamundi, 1997]. La subsidence totale du Carbonifère (courbe SUTO, fig. 3A) est d’environ 750 m et la subsidence tectonique sous eau (courbe SUTE, fig. 3A) est d’environ la moitié de cette valeur. Deux phases de subsidence sont identifiables : la première du Tournaisien au Bashkirien inférieur avec un faible taux de subsidence tectonique (2 m/Ma) et la seconde du Bashkirien supérieur à l’Assélien avec un taux un peu plus important de subsidence tectonique (22 m/Ma). La méthode proposée par Middleton [1980] pour les bassins intracratoniques américains fut utilisée pour modéliser la subsidence tectonique observée dans le MB (fig. 3B). Le modèle est caractérisé par une phase de subsidence initiale plus faible pendant la période de chauffage de la lithosphère que lors de la seconde phase de subsidence thermique pendant le refroidissement de la lithosphère. Le rapport entre la variation eustatique du niveau marin (DSLE, fig. 3A) et la subsidence tectonique à l’air libre (SUAL) permet de calculer le rapport eustatisme/ tectonique (E/T) qui est égal à 4 en faveur de l’eustatisme pendant la première phase et à 0,3 à l’avantage de la tectonique pendant la deuxième phase. La phase de subsidence 1 correspond aux séquences du second ordre D, 0 et I et la phase 2 aux séquences II à VII. Le bassin du Dniepr-Donets (DDB). – Le DDB est un rift situé entre deux massifs précambriens et est divisé en différents segments, appelés Pripyat, Dniepr, Donets et Donbass (fig. 1). Le DDB présente environ 14 km de sédiments principalement terrigènes dans le segment du Dniepr et environ 21 km dans le Donets et Donbass, d’âge dévonien moyen à sakmarien [Izart et al., 1996 ; 1998]. La couverture d’âge mésozoïque et cénozoïque a une épaisseur de 2 km excepté dans le Donbass où elle a été érodée. Une subsidence tectonique maximale d’environ 3,4 km fut calculée dans le Dniepr par van Wees et al., [1996]. Selon ces auteurs, le segment du Dniepr présente une phase de rifting initiale pendant le Dévonien supérieur et une phase post-rifting du Carbonifère inférieur à la base du Mésozoïque avec quelques rajeunissements, suivie par une inversion tectonique. A la limite entre le segment du Donets et du Donbass nous avons calculé une subsidence totale de 22,8 km et une subsidence tectonique d’environ 6,1 km (fig. 3C). Deux phases tectoniques peuvent être distinguées : la première du Dévonien au Carbonifère inférieur qui présente un taux moyen de subsidence tectonique de 40 m/Ma correspond à la phase du rifting initial et du début de la phase post-rifting du Dniepr et la seconde phase de rifting du Viséen supérieur à l’Assélien qui présente un taux important de subsidence tectonique de 90 m/Ma correspond aux rajeunissements du segment du Dniepr. Un soulèvement a lieu au Sakmarien, puis une compression pendant le Trias supérieur et à la limite Crétacé-Tertiaire [Stovba et Stephenson, 1999]. La subsidence tectonique fut modélisée (fig. 3D) en utilisant la méthode de Royden et Keen [1980]. Les deux phases tectoniques, appelées rifting 1 et 2, furent modélisées successivement. Les facteurs d’extension crustale (δ) sont respectivement pour les deux phases de 1,18 et 3,5 et les facteurs d’extension sous-crustale (β) de 1,1. Le rapport E/T est de 0,24 en faveur de la tectonique pendant la phase 1 et de 0,03 pendant la phase 2. La phase 1 correspond aux séquences du second ordre D et 0 et la phase 2 aux séquences I à VII (fig. 2). Les segments du Dniepr, Donets et Donbass possèdent donc les mêmes caractéristiques tectoniques, avec une intensité plus importante dans le Donets et le Donbass. Conclusion. – Le rifting d’âge dévonien supérieur a existé dans le DDB et probablement aussi dans le MB. L’histoire de ces deux bassins diverge ensuite avec la poursuite du rifting dans le seul DDB. Le MB est un bassin intracratonique qui peut être modélisé avec une phase de chauffage du Dévonien au Bashkirien et une phase de refroidissement engendrant une subsidence thermique du Moscovien à l’Assélien. Le DDB est un rift montrant une première phase de rifting durant le Dévonien supérieur, une phase post-rift jusqu’au Viséen supérieur et une deuxième phase de rifting jusqu’à l’Assélien uniquement dans les segments du Donets et Donbass. Si l’eustatisme contrôle la sédimentation dans le MB, la tectonique prévaut dans le DDB

The Economic Advantage: Assessing the value of climate-change actions in agriculture

This report is aimed at readers who seek to build economic evidence in support of the inclusion of actions on agriculture in climate change plans and programmes, particularly at the national level under the umbrella of nationally determined contributions (NDCs) to the December 2015 Paris Agreement, which aims to restrict a rise in global temperatures and manage risks

Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans