Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer's disease (AD). We have previously shown that hypoxia in vitro alters Ca2+ homeostasis in astrocytes and promotes increased production of amyloid beta peptides (Abeta) of AD. Indeed, alteration of Ca2+ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O2, 24h) in a manner that is independent of amyloid beta peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid beta peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either beta or gamma secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Abeta production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.

Hypoxia suppresses glutamate transport in astrocytes

Glutamate uptake by astrocytes is fundamentally important in the regulation of CNS function. Disruption of uptake can lead to excitotoxicity and is implicated in various neurodegenerative processes as well as a consequence of hypoxic/ischemic events. Here, we investigate the effect of hypoxia on activity and expression of the key glutamate transporters excitatory amino acid transporter 1 (EAAT1) [GLAST (glutamate-aspartate transporter)] and EAAT2 [GLT-1 (glutamate transporter 1)]. Electrogenic, Na+-dependent glutamate uptake was monitored via whole-cell patch-clamp recordings from cortical astrocytes. Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake was significantly reduced, and pharmacological separation of uptake transporter subtypes suggested that the EAAT2 subtype was preferentially reduced relative to the EAAT1. This suppression was confirmed at the level of EAAT protein expression (via Western blots) and mRNA levels (via real-time PCR). These effects of hypoxia to inhibit glutamate uptake current and EAAT protein levels were not replicated by desferrioxamine, cobalt, FG0041, or FG4496, agents known to mimic effects of hypoxia mediated via the transcriptional regulator, hypoxia-inducible factor (HIF). Furthermore, the effects of hypoxia were not prevented by topotecan, which prevents HIF accumulation. In stark contrast, inhibition of nuclear factor-kappaB (NF-kappaB) with SN50 fully prevented the effects of hypoxia on glutamate uptake and EAAT expression. Our results indicate that prolonged hypoxia can suppress glutamate uptake in astrocytes and that this effect requires activation of NF-kappaB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

Identifying time lags in the restoration of grassland butterfly communities: a multi-site assessment

Although grasslands are crucial habitats for European butterflies, large-scale declines in quality and area have devastated many species. Grassland restoration can contribute to the recovery of butterfly populations, although there is a paucity of information on the long-term effects of management. Using eight UK data sets (9-21 years), we investigate changes in restoration success for (1) arable reversion sites, were grassland was established on bare ground using seed mixtures, and (2) grassland enhancement sites, where degraded grasslands are restored by scrub removal followed by the re-instigation of cutting/grazing. We also assessed the importance of individual butterfly traits and ecological characteristics in determining colonisation times. Consistent increases in restoration success over time were seen for arable reversion sites, with the most rapid rates of increase in restoration success seen over the first 10 years. For grasslands enhancement there were no consistent increases in restoration success over time. Butterfly colonisation times were fastest for species with widespread host plants or where host plants established well during restoration. Low mobility butterfly species took longer to colonise. We show that arable reversion is an effective tool for the management of butterfly communities. We suggest that as restoration takes time to achieve, its use as a mitigation tool against future environmental change (i.e. by decreasing isolation in fragmented landscapes) needs to take into account such time lags.

Novel management to enhance spider biodiversity in existing grass buffer strips

Grass buffer strips have been widely sown to mitigate against intensive agricultural management practices that have negatively impacted on invertebrate and plant biodiversity in arable farming systems. Typically, such strips are floristically species poor and are dominated by grasses. In the present study, we developed management practices to enhance the floristic and structural diversity of these existing strips for the benefit of spiders, a key provider of natural pest control in crops. Across three UK arable farms, we investigated the benefits of: (i) scarification to create germination niches into which wildflower seeds were sown and (ii) the effect of graminicide applications to suppress grass dominance. Spiders were sampled twice per year (July and September) during 2008 and 2009. The combination of scarification with wildflower seeds, as well as graminicide, resulted in the greatest wildflower cover and lowest grass cover, with a general trend of increased abundance of adult and juvenile spiders. The abundance of Pachygnatha degeeri, Bathyphantes gracilis and juvenile wolf spiders of the genus Pardosa was positively correlated with wildflower cover, probably reflecting increased prey availability. Sward structure was negatively correlated with Erigone atra, Oedothorax fuscus and juvenile Pardosa abundance. Management that utilizes existing commonly adopted agri-environment options, such as grass buffer strips, represents a potentially important conservation tool for increasing the quantity and quality of invertebrate habitats. This can maximize opportunities for the provision of multiple ecosystem services, including pest regulation by predators such as spiders. These management practices have the potential to be incorporated into existing U.K. and European agri-environment schemes.

Enhancement of buffer strips can improve provision of multiple ecosystem services

Farmland invertebrates play a pivotal role in the provision of ecosystem services, i.e. services that benefit humans. For example, bumblebees, solitary bees and honeybees, are crucial to the pollination of many of the world's crops and wildflowers, with over 70% of the world's major food crops dependent on the pollination services provided by these insects. The larvae of some butterfly species are considered to be pests; however, together with moth and sawfly larvae, they represent a key dietary component for many farmland birds. Spiders and ground beetles predate on crop pests including aphids, whilst soil macrofauna such as earthworms are vital for soil fertility services and nutrient recycling. Despite their importance, population declines of invertebrates have been observed during the last sixty years in the UK and NW Europe. For example, seven UK bumblebee species are in decline, and in the last 20 years, the species Bombus subterraneus (short-haired bumblebee) has become extinct, whilst there was a 54% decline in honeybee colony numbers in England from 1985 to 2005. Comparable trends have been documented for butterflies with a 23% decline in UK farmland species such as Anthocharis cardamines (orange tip) between 1990 and 2007. These declines have been widely attributed to the modern intensive arable management practices that have been developed to maximise crop yield. For example, loss and fragmentation of foraging and nesting habitats, including species-rich meadows and hedgerows, have been implicated in the decline of bees and butterflies. Increased use of herbicides and fertilisers has caused detrimental effects on many plant species with negative consequences for predatory invertebrates such as spiders and beetles which rely on plants for food and shelter.

Maternally expressed gene1 is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.

I see happy people: attention bias towards happy but not angry facial expressions in Williams syndrome

Introduction: Observations of behaviour and research using eye-tracking technology have shown that individuals with Williams syndrome (WS) pay an unusual amount of attention to other people’s faces. The present research examines whether this attention to faces is moderated by the valence of emotional expression. Method: Sixteen participants with WS aged between 13 and 29 years (Mean=19 years 9 months) completed a dot-probe task in which pairs of faces displaying happy, angry and neutral expressions were presented. The performance of the WS group was compared to two groups of typically developing control participants, individually matched to the participants in the WS group on either chronological age or mental age. General mental age was assessed in the WS group using the Woodcock Johnson Test of Cognitive Ability Revised (WJ-COG-R; Woodcock & Johnson, 1989; 1990). Results: Compared to both control groups, the WS group exhibited a greater attention bias for happy faces. In contrast, no between-group differences in bias for angry faces were obtained. Conclusions: The results are discussed in relation to recent neuroimaging findings and the hypersocial behaviour that is characteristic of the WS population.

Modelling the effects of gravity waves on stratocumulus clouds observed during VOCALS-UK

During the VOCALS campaign spaceborne satellite observations showed that travelling gravity wave packets, generated by geostrophic adjustment, resulted in perturbations to marine boundary layer (MBL) clouds over the south-east Pacific Ocean (SEP). Often, these perturbations were reversible in that passage of the wave resulted in the clouds becoming brighter (in the wave crest), then darker (in the wave trough) and subsequently recovering their properties after the passage of the wave. However, occasionally the wave packets triggered irreversible changes to the clouds, which transformed from closed mesoscale cellular convection to open form. In this paper we use large eddy simulation (LES) to examine the physical mechanisms that cause this transition. Specifically, we examine whether the clearing of the cloud is due to (i) the wave causing additional cloud-top entrainment of warm, dry air or (ii) whether the additional condensation of liquid water onto the existing drops and the subsequent formation of drizzle are the important mechanisms. We find that, although the wave does cause additional drizzle formation, this is not the reason for the persistent clearing of the cloud; rather it is the additional entrainment of warm, dry air into the cloud followed by a reduction in longwave cooling, although this only has a significant effect when the cloud is starting to decouple from the boundary layer. The result in this case is a change from a stratocumulus to a more patchy cloud regime. For the simulations presented here, cloud condensation nuclei (CCN) scavenging did not play an important role in the clearing of the cloud. The results have implications for understanding transitions between the different cellular regimes in marine boundary layer (MBL) clouds.

The effect of artificial diet on the production of alarm pheromone by Myzus persicae.

The cornicle secretion of Myzus persicae reared on artificial diet only elicits an alarm response in plant-reared conspecifics after the young aphids have been transferred to plants for 7days. Acetate in the form of 0.32% sodium acetate has been added to the diet as an early step in synthesis of the alarm pheromone, (E)-β-farnesene (EBF). The cornicle secretion of diet-reared aphids then elicits an alarm response. However, there is no difference in internal EBF concentration between plant- and diet-reared aphids. Puncturing aphids, either plant- or diet-reared, with a pin shows that both can elicit an alarm response, whereas it is reduced by half with diet-reared individuals. Although there is no significant difference in the concentration of EBF produced, the total amount in diet-reared aphids is increased by acetate in the diet to a level similar to that in plant-reared individuals: the size of aphids reared on an acetate-supplemented diet is increased and comparable with the size of those that are plant-reared. Bioassays with a range of EBF concentrations show a high threshold for the alarm response. It is concluded that the different size of aphids reared on plants and standard diet results in them secreting, respectively, above and below the response threshold.

The DACCIWA project: dynamics-aerosol-chemistry-cloud interactions in West Africa

Massive economic and population growth, and urbanization are expected to lead to a tripling of anthropogenic emissions in southern West Africa (SWA) between 2000 and 2030. However, the impacts of this on human health, ecosystems, food security, and the regional climate are largely unknown. An integrated assessment is challenging due to (a) a superposition of regional effects with global climate change, (b) a strong dependence on the variable West African monsoon, (c) incomplete scientific understanding of interactions between emissions, clouds, radiation, precipitation, and regional circulations, and (d) a lack of observations. This article provides an overview of the DACCIWA (Dynamics-Aerosol-Chemistry-Cloud Interactions in West Africa) project. DACCIWA will conduct extensive fieldwork in SWA to collect high-quality observations, spanning the entire process chain from surface-based natural and anthropogenic emissions to impacts on health, ecosystems, and climate. Combining the resulting benchmark dataset with a wide range of modeling activities will allow (a) assessment of relevant physical, chemical, and biological processes, (b) improvement of the monitoring of climate and atmospheric composition from space, and (c) development of the next generation of weather and climate models capable of representing coupled cloud-aerosol interactions. The latter will ultimately contribute to reduce uncertainties in climate predictions. DACCIWA collaborates closely with operational centers, international programs, policy-makers, and users to actively guide sustainable future planning for West Africa. It is hoped that some of DACCIWA’s scientific findings and technical developments will be applicable to other monsoon regions.

Half-remembered quotations from mostly forgotten speeches: the limits of Labour’s European policy discourse

This article uses discourse analysis to study the continuities in British foreign policy thinking within the Labour party from the 1960s to the present day. Using representative extracts from speeches by Hugh Gaitskell, Harold Wilson, Tony Blair and Gordon Brown, it identifies the ideational consis- tencies in the leaders’ attitudes to: Empire; federalism in the EEC/EU; and laying down conditions that have to be met before any constructive engagement with ‘Europe’ can be countenanced. We argue that these consistencies, spanning a 50-year period, exemplify a certain stagnation both within Labour’s European discourses and within British foreign policy thinking more widely. We develop the idea that Labour party thinking has been crucially framed by both small ‘c’ conser- vative and upper-case Conservative ideology, popularised by Winston Churchill in his ‘three circles’ model of British foreign policy.

The climate and environment of Byzantine Anatolia: integrating science, history, and archaeology

The integration of high-resolution archaeological, textual, and environmental data with longer-term, low-resolution data affords greater precision in identifying some of the causal relationships underlying societal change. Regional and microregional case studies about the Byzantine world—in particular, Anatolia, which for several centuries was the heart of that world—reveal many of the difficulties that researchers face when attempting to assess the influence of environmental factors on human society. The Anatolian case challenges a number of assumptions about the impact of climatic factors on socio-political organization and medium-term historical evolution, highlighting the importance of further collaboration between historians, archaeologists, and climate scientists.

Local and landscape effects on bee functional guilds in pigeon pea crops in Kenya

Pollinators face many challenges within agricultural systems due to landscape changes and intensification which can affect resource availability that can impact pollination services. This paper examines pigeon pea pollination and considers how landscape context and agricultural intensification in terms of pesticide use affects the abundance of bees characterized by species guilds on crops. The study was conducted on six paired farms across a gradient of habitat complexity based on the distance of each farm from adjacent semi-natural vegetation in Kibwezi Sub-county, Kenya. The study found that farms which do not use insecticides in farm management, but are in close proximity to natural habitat have greater bee guild abundance, but at further distances, overall abundance is reduced with or without insecticide use. At 1 km landscape radius, the complexity of habitats but not patch size had a positive impact on the abundance of cavity nesting bees and mason bees, which can be attributed to the interspersion of the small-holder farms with semi-natural habitats across the landscapes producing mosaics of heterogeneous habitats. The study revealed the strongest relationships between fruit set and bee abundance to be with the carpenter bee, social bee and solitary bee guilds, which are among the most abundant bees visiting pigeon pea flowers in this system. Our findings provide the foundation for conservation efforts by identifying which bee guilds pollinated pigeon peas. From this study, we suggest managing the floral and nesting resources that would best support the most abundant crop pollinators, and also reducing insecticide application to the crop.

Social and ecological drivers of success in agri-environment schemes: the roles of farmers and environmental context

1. Agri-environment schemes remain a controversial approach to reversing biodiversity losses, partly because the drivers of variation in outcomes are poorly understood. In particular, there is a lack of studies that consider both social and ecological factors. 2. We analysed variation across 48 farms in the quality and biodiversity outcomes of agri-environmental habitats designed to provide pollen and nectar for bumblebees and butterflies or winter seed for birds. We used interviews and ecological surveys to gather data on farmer experience and understanding of agri-environment schemes, and local and landscape environmental factors. 3. Multimodel inference indicated social factors had a strong impact on outcomes and that farmer experiential learning was a key process. The quality of the created habitat was affected positively by the farmer’s previous experience in environmental management. The farmer’s confidence in their ability to carry out the required management was negatively related to the provision of floral resources. Farmers with more wildlife-friendly motivations tended to produce more floral resources, but fewer seed resources. 4. Bird, bumblebee and butterfly biodiversity responses were strongly affected by the quantity of seed or floral resources. Shelter enhanced biodiversity directly, increased floral resources and decreased seed yield. Seasonal weather patterns had large effects on both measures. Surprisingly, larger species pools and amounts of semi-natural habitat in the surrounding landscape had negative effects on biodiversity, which may indicate use by fauna of alternative foraging resources. 5. Synthesis and application. This is the first study to show a direct role of farmer social variables on the success of agri-environment schemes in supporting farmland biodiversity. It suggests that farmers are not simply implementing agri-environment options, but are learning and improving outcomes by doing so. Better engagement with farmers and working with farmers who have a history of environmental management may therefore enhance success. The importance of a number of environmental factors may explain why agri-environment outcomes are variable, and suggests some – such as the weather – cannot be controlled. Others, such as shelter, could be incorporated into agri-environment prescriptions. The role of landscape factors remains complex and currently eludes simple conclusions about large-scale targeting of schemes.