Individuals within species frequently show differences in traits. Yet, until relatively recently, researchers treated all conspecifics as ecologically equivalent. I investigated how environmental variation, temperature and sex may influence intraspecific variation in behavioural, morphological and physiological traits, and their covariance, in the ground beetle Carabus hortensis - a species that is currently undergoing range expansion.
By examining intraspecific variation in morphological traits across the C. hortensis expansion front, I showed that male, but not female, body size increased with proximity to the range edge. This may suggest that males evolved larger bodies and longer legs to increase mate searching efficiency where female density is low. Secondly, I found intraspecific variation in C. hortensis thermal biology, with males being active over a wider range of temperatures than females, and large females being more thermally sensitive than smaller females. Additionally, I showed that male and female movement in the wild is differentially influenced by temperature, and that laboratory measures of animal personality differences can be predictive of intraspecific variation in movement patterns in the wild.
I then tested the relationships between metabolic rate, exploratory behaviour and morphology, finding that the strength, direction, and temperature dependency of relationships differed between the sexes. Finally, I demonstrated that that relationships between metabolic rate and body mass are uninfluenced by temperature, and that individuals with high average metabolic rates and exploratory behaviour are more thermally sensitive.
My findings provide new insights into the roles of sex and thermal sensitivity in shaping intraspecific variation in traits, and their implications for individual fitness and population dynamics under continued climate change. Overall, my results suggest that increasing temperatures may select for smaller individuals and those with lower average metabolic rates and exploratory behaviour.

Hsp90 (Heat Shock Protein 90) is an ATP (Adenosine triphosphate) molecular chaperone responsible for the activation and maturation of client proteins. The mechanism by which Hsp90 achieves such activation, involving structurally diverse client proteins, has remained enigmatic. However, recent advances using structural techniques, together with advances in biochemical studies, have not only defined the chaperone cycle but have shed light on its mechanism of action. Hsp90 hydrolysis of ATP by each protomer may not be simultaneous and may be dependent on the specific client protein and co-chaperone complex involved. Surprisingly, Hsp90 appears to remodel client proteins, acting as a means by which the structure of the client protein is modified to allow its subsequent refolding to an active state, in the case of kinases, or by making the client protein competent for hormone binding, as in the case of the GR (glucocorticoid receptor). This review looks at selected examples of client proteins, such as CDK4 (cyclin-dependent kinase 4) and GR, which are activated according to the so-called ‘remodelling hypothesis’ for their activation. A detailed description of these activation mechanisms is paramount to understanding how Hsp90-associated diseases develop.

Inframe insertion and deletion mutations (indels) are commonly observed in cancer samples accounting for over 1% of all reported mutations. Few somatic inframe indels have been clinically documented as pathogenic and at present there are few tools to predict which indels drive cancer development. However, indels are a common feature of hereditary disease and several tools have been developed to predict the impact of inframe indels on protein function. In this study, we test whether six of the popular prediction tools can be adapted to test for cancer driver mutations and then develop a new algorithm (IndelRF) that discriminates between recurrent indels in known cancer genes and indels not associated with disease. IndelRF was developed to try and identify somatic, driver, and inframe indel mutations. Using a random forest classifier with 11 features, IndelRF achieved accuracies of 0.995 and 0.968 for insertion and deletion mutations, respectively. Finally, we use IndelRF to classify the inframe indel cancer mutations in the MOKCa database.

Abrupt land change, such as deforestation or agricultural intensification, is a key driver of biodiversity change. Following abrupt land change, local biodiversity often continues to be influenced through biotic lag effects. However, current understanding of how terrestrial biodiversity is impacted by past abrupt land changes is incomplete. Here we show that abrupt land change in the past continues to influence present species assemblages globally. We combine geographically and taxonomically broad data on local biodiversity with quantitative estimates of abrupt land change detected within time series of satellite imagery from 1982 to 2015. Species richness and abundance were 4.2% and 2% lower, respectively, and assemblage composition was altered at sites with an abrupt land change compared to unchanged sites, although impacts differed among taxonomic groups. Biodiversity recovered to levels comparable to unchanged sites after >10 years. Ignoring delayed impacts of abrupt land changes likely results in incomplete assessments of biodiversity change.

The Hsp90 co-chaperone R2TP consists of the AAA+ ATPases, RUVBL1 (Rvb1p in yeast) and RUVBL2 (Rvb2 in yeast), which together make up a heterohexameric ring, in complex with PIH1D1 (Pih1p in yeast) and RPAP3 (Tah1p in yeast). R2TP is involved in the activation of client proteins, such as phosphatidylinositol 3 kinase-related kinases, including mTORC1, ATM, DNA-PK, SMG and ATR/ATRIP, or in the assembly of protein complexes including those of RNA polymerase and snoRNPs, amongst others. In other cases, the role of the TP component (RPAP3-PIH1D1) of R2TP, and consequently Hsp90, is controversial. None-the-less, the extensive role of RUVBL1-RUVBL2 complex in cells, either with or without Hsp90, means that dysfunction of these AAA+ ATPases, Hsp90 or components of the complexes they assemble leads to diseases such as cancer, ciliary dyskinesia and in the case of defects in ATM to ataxia telangiectasia-like syndrome. Recent advances in determining the structure of the R2TP complex has led to an increased understanding of the assembly and function of the R2TP complex. In this review we discuss the current structural advances in determining the architecture of the R2TP complex and the advances made in understanding its active state.

Complex conformational dynamics are essential for function of the dimeric molecular cha- perone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimer- ization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

Human use of the land (for agriculture and settlements) has a substantial negative effect on biodiversity globally. However, not all species are adversely affected by land use, and indeed, some benefit from the creation of novel habitat. Geographically rare species may be more negatively affected by land use than widespread species, but data limitations have so far prevented global multi-clade assessments of land-use effects on narrow-ranged and widespread species. We analyse a large, global database to show consistent differences in assemblage composition. Compared with natural habitat, assemblages in disturbed habitats have more widespread species on average, especially in urban areas and the tropics. All else being equal, this result means that human land use is homogenizing assemblage composition across space. Disturbed habitats show both reduced abundances of narrow-ranged species and increased abundances of widespread species. Our results are very important for biodiversity conservation because narrow-ranged species are typically at higher risk of extinction than widespread species. Furthermore, the shift to more widespread species may also affect ecosystem functioning by reducing both the contribution of rare species and the diversity of species’ responses to environmental changes among local assemblages.

Most land on Earth has been changed by humans and past changes of land can have lasting influences on current species assemblages. Yet few globally representative studies explicitly consider such influences even though auxiliary data, such as from remote sensing, are readily available. Time series of satellite-derived data have been commonly used to quantify differences in land-surface attributes such as vegetation cover, which will among other things be influenced by anthropogenic land conversions and modifications. Here we quantify differences in current and past (up to five years before sampling) vegetation cover, and assess whether such differences differentially influence taxonomic and functional groups of species assemblages between spatial pairs of sites. Specifically, we correlated between-site dissimilarity in photosynthetic activity of vegetation (the Enhanced Vegetation Index) with the corresponding dissimilarity in local species assemblage composition from a global database using a common metric for both, the Bray-Curtis index. We found that dissimilarity in species assemblage composition was on average more influenced by dissimilarity in past than current photosynthetic activity, and that the influence of past dissimilarity increased when longer time periods were considered. Responses to past dissimilarity in photosynthetic activity also differed among taxonomic groups (plants, invertebrates, amphibians, reptiles, birds and mammals), with reptiles being among the most influenced by more dissimilar past photosynthetic activity. Furthermore, we found that assemblages dominated by smaller and more vegetation-dependent species tended to be more influenced by dissimilarity in past photosynthetic activity than prey-dependent species. Overall, our results have implications for studies that investigate species responses to current environmental changes and highlight the importance of past changes continuing to influence local species assemblage composition. We demonstrate how local species assemblages and satellite-derived data can be linked and provide suggestions for future studies on how to assess the influence of past environmental changes on biodiversity.

Bi-stable perception has been an important tool to investigate how visual input is interpreted and how it reaches consciousness. To explain the mechanisms of this phenomenon, it has been assumed that a mutual inhibition circuit plays a key role. It is possible that this circuit functions to resolve ambiguity of input image by quickly shifting the balance of competing signals in response to conflicting features. Recently we established an in vitro neural recording system combined with computerized connections mediated by model neurons and synapses (“dynamic clamp” system). With this system, mutual inhibition circuit between two pyramidal cells from primary visual cortex were established by model inhibitory neurons and model synapses. Simultaneous injection of depolarizing current to the two pyramidal cells caused bi-stable activities: dominance of neural activities alternated between the two neurons with an interval of several seconds. We report the effect of adding noise to the (real) pyramidal cells and the (model) inhibitory neurons. Both excitatory and inhibitory synaptic conductance noise was modelled and given to these neurons while the pyramidal cells were exhibiting bi-stable activity. The histogram of the dominant activity durations showed gamma-like skewed distributions. The skewedness was enhanced by increasing the standard deviation of the conductance noise and the durations decreased overall. While adaptation of the dominant neuron and recovery (from adaptation) of the suppressed neuron caused a decrease and increase of their excitabilities, the fluctuation of membrane potentials due to the given conductance noise appeared to facilitate the reversal of the dominance.

Sporting a mix of blue, yellow, white, green and black, the unmistakable Blue Tit reflects the colours of a planet affected by a burgeoning human population. Fortunately, Blue Tits are adapting well to modern humanity, taking advantage of our propensity to feed birds in our gardens and provide boxes for them to nest in. In turn, this feisty little species provides an excellent model for biological research. This book is the result of a personal quest by author Martyn Stenning to bring together a range of discoveries into one accessible volume.

The Blue Tit begins by inviting readers into the intimate lives of these birds as they attempt to reproduce, describing the many challenges they face when rearing their offspring. The story moves on to the fluid state of Blue Tit classification across the native Palearctic range, before progressing into population structure, lifetime ecology and an exploration of factors that determine breeding success. It culminates with an in-depth look at research over the years, followed by a selection of personal anecdotes and an overview of Blue Tit appearances in folklore and poetry.

This book provides a definitive record of the biology and ecology of one of our most popular, intelligent and charismatic birds.

The PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems) has collated ecological survey data from hundreds of published biodiversity comparisons of sites facing different land-use and related pressures, and used the resulting taxonomically and geographically broad database (abundance and occurrence data for over 50,000 species and over 30,000 sites in nearly 100 countries) to develop global biodiversity models, indicators and projections. After outlining the science and science-policy gaps that motivated PREDICTS, this review discusses the key design decisions that helped it to achieve its objectives. In particular, we discuss basing models on a large, taxonomically and geographically representative database, so that they may be applicable to biodiversity more broadly; space-for-time substitution, which allows estimation of pressure-state models without the need for representative time-series data; and collation of raw data rather than statistical results, greatly expanding the range of response variables that can be modelled. The heterogeneity of data in the PREDICTS database has presented a range of modelling challenges: we discuss these with a focus on our implementation of the Biodiversity Intactness Index, an indicator with considerable policy potential but which had not previously been estimated from primary biodiversity data. We then summarise the findings from analyses of how land use and related pressures affect local () diversity and spatial turnover ( diversity), and how these effects are mediated by ecological attributes of species. We discuss the relevance of our findings for policy, before ending with some directions of ongoing and possible future research.

An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.

Alternative polyadenylation (APA) is a widespread gene regulatory mechanism that generates mRNAs with different 3′-ends, allowing them to interact with different sets of RNA regulators such as microRNAs and RNA-binding proteins. Recent studies have shown that during development, neural tissues produce mRNAs with particularly long 3′UTRs, suggesting that such extensions might be important for neural development and function. Despite this, the mechanisms underlying neural APA are not well understood. Here, we investigate this problem within the Drosophila nervous system, focusing on the roles played by general cleavage and polyadenylation factors (CPA factors). In particular, we examine the model that modulations in CPA factor concentration may affect APA during development. For this, we first analyse the expression of the Drosophila orthologues of all mammalian CPA factors and note that their expression decreases during embryogenesis. In contrast to this global developmental decrease in CPA factor expression, we see that cleavage factor I (CFI) expression is actually elevated in the late embryonic central nervous system, suggesting that CFI might play a special role in neural tissues. To test this, we use the UAS/Gal4 system to deplete CFI proteins from neural tissue and observe that in this condition, multiple genes switch their APA patterns, demonstrating a role of CFI in APA control during Drosophila neural development. Furthermore, analysis of genes with 3′UTR extensions of different length leads us to suggest a novel relation between 3′UTR length and sensitivity to CPA factor expression. Our work thus contributes to the understanding of the mechanisms of APA control within the developing central nervous system.

Land-use change is the single biggest driver of biodiversity loss in the tropics. Biodiversity models can be useful tools to inform policymakers and conservationists of the likely response of species to anthropogenic pressures, including land-use change. However, such models generalize biodiversity responses across wide areas and many taxa, potentially missing important characteristics of particular sites or clades. Comparisons of biodiversity models with independently collected field data can help us understand the local factors that mediate broad-scale responses. We collected independent bird occurrence and abundance data along two elevational transects in Mount Kilimanjaro, Tanzania and the Taita Hills, Kenya. We estimated the local response to land use and compared our estimates with modelled local responses based on a large database of many different taxa across Africa. To identify the local factors mediating responses to land use, we compared environmental and species assemblage information between sites in the independent and African-wide datasets. Bird species richness and abundance responses to land use in the independent data followed similar trends as suggested by the African-wide biodiversity model, however the land-use classification was too coarse to capture fully the variability introduced by local agricultural management practices. A comparison of assemblage characteristics showed that the sites on Kilimanjaro and the Taita Hills had higher proportions of forest specialists in croplands compared to the Africa-wide average. Local human population density, forest cover and vegetation greenness also differed significantly between the independent and Africa-wide datasets. Biodiversity models including those variables performed better, particularly in croplands, but still could not accurately predict the magnitude of local species responses to most land uses, probably because local features of the land management are still missed. Overall, our study demonstrates that local factors mediate biodiversity responses to land use and cautions against applying biodiversity models to local contexts without prior knowledge of which factors are locally relevant.

Agricultural conversion of tropical forests is a major driver of biodiversity loss. Slowing rates of deforestation is a conservation priority, but it is also useful to consider how species diversity is retained across the agricultural matrix. Here we assess how bird diversity varies in relation to land use in the Taita Hills, Kenya. We used point counts to survey birds along a land-use gradient that included primary forest, secondary vegetation, agroforest, timber plantation and cropland. We found that the agricultural matrix supports an abundant and diverse bird community with high levels of species turnover, but that forest specialists are confined predominantly to primary forest, with the matrix dominated by forest visitors. Ordination analyses showed that representation of forest specialists decreases with distance from primary forest. With the exception of forest generalists, bird abundance and diversity are lowest in timber plantations. Contrary to expectation, we found feeding guilds at similar abundances in all land-use types. We conclude that while the agricultural matrix, and agroforest in particular, makes a strong contribution to observed bird diversity at the landscape scale, intact primary forest is essential for maintaining this diversity, especially among species of conservation concern.

As part of a study into the molecular genetics of sexually dimorphic complex traits, we used high-throughput sequencing to obtain data on genomic variation in an outbred laboratory-adapted fruit fly (Drosophila melanogaster) population. We successfully resequenced the whole genome of 220 hemiclonal females that were heterozygous for the same Berkeley reference line genome (BDGP6/dm6), and a unique haplotype from the outbred base population (LHM). The use of a static and known genetic background enabled us to obtain sequences from whole-genome phased haplotypes. We used a BWA-Picard-GATK pipeline for mapping sequence reads to the dm6 reference genome assembly, at a median depth-of coverage of 31X, and have made the resulting data publicly-available in the NCBI Short Read Archive (Accession number SRP058502). We used Haplotype Caller to discover and genotype 1,726,931 small genomic variants (SNPs and indels, <200bp). Additionally we detected and genotyped 167 large structural variants (1-100Kb in size) using GenomeStrip/2.0. Sequence and genotype data are publicly-available at the corresponding NCBI databases: Short Read Archive, dbSNP and dbVar (BioProject PRJNA282591). We have also released the unfiltered genotype data, and the code and logs for data processing and summary statistics.

In a complex organism, cell proliferation and apoptosis need to be precisely controlled in order for tissues to develop correctly. Excessive cell proliferation can lead to diseases such as cancer. We have shown that the exoribonuclease Dis3L2 is required for the correct regulation of proliferation in a natural tissue within the model organism Drosophila melanogaster. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3′-5′ direction. We show that knockdown of dis3L2 in the Drosophila wing imaginal discs results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq we identified a small set of mRNAs that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, we identified 2 that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). We also demonstrate a compensatory effect between Dis3L2 and the 5′-3′ exoribonuclease Pacman demonstrating that these 2 exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome.

Oligodendrocytes are produced from the same region of the ventral spinal cord that earlier generated motor neurons in bird and rodent embryos. Motor neuron and oligodendrocyte precursor cells express Olig genes, which encode basic helix-loop-helix transcription factors that play important roles in the development of both motor neurons and oligodendrocytes. We found that oligodendrocytes develop similarly in zebrafish embryos, in that they arise from ventral spinal cord and migrate to new positions. Developing primary motor neurons and oligodendrocytes express olig2 as do neural plate cells that give rise to both primary motor neurons and oligodendrocytes. Loss of olig2 function prevented primary motor neuron and oligodendrocyte development, whereas olig2 overexpression promoted formation of excess primary motor neurons and oligodendrocytes. We provide genetic evidence that Hedgehog signaling is required for zebrafish olig2 expression and oligodendrocyte development. However, olig2 overexpression did not promote primary motor neuron or oligodendrocyte development in embryos with reduced Hedgehog signaling activity. One possibility consistent with these data is that Hedgehog signaling, partly by inducing olig2 expression, specifies neural precursor cells that have potential for primary motor neuron or oligodendrocyte fate

Systemic inflammation in humans may be triggered by infection, termed sepsis, or non-infective processes, termed non-infective systemic inflammatory response syndrome (SIRS). MicroRNAs regulate cellular processes including inflammation and may be detected in blood. We aimed to establish definitive proof-of-principle that circulating microRNAs are differentially affected during sepsis and non-infective SIRS. Critically ill patients with severe (n = 21) or non-severe (n = 8) intra-abdominal sepsis; severe (n = 23) or non-severe (n = 21) non-infective SIRS; or no SIRS (n = 16) were studied. Next-generation sequencing and qRT-PCR were used to measure plasma microRNAs. Detectable blood miRNAs (n = 116) were generally up-regulated in SIRS compared to no-SIRS patients. Levels of these 'circulating inflammation-related microRNAs' (CIR-miRNAs) were 2.64 (IQR: 2.10-3.29) and 1.52 (IQR: 1.15-1.92) fold higher for non-infective SIRS and sepsis respectively (p < 0.0001), hence CIR-miRNAs appeared less abundant in sepsis than in SIRS. Six CIR-miRNAs (miR-30d-5p, miR-30a-5p, miR-192-5p, miR-26a-5p, miR-23a-5p, miR-191-5p) provided good-to-excellent discrimination of severe sepsis from severe SIRS (0.742-0.917 AUC of ROC curves). CIR-miRNA levels inversely correlated with pro-inflammatory cytokines (IL-1, IL-6 and others). Thus, among critically ill patients, sepsis and non-infective SIRS are associated with substantial, differential changes in CIR-miRNAs. CIR-miRNAs may be regulators of inflammation and warrant thorough evaluation as diagnostic and therapeutic targets.

The late Quaternary megafauna extinction was a severe global-scale event. Two factors, climate change and modern humans, have received broad support as the primary drivers, but their absolute and relative importance remains controversial. To date, focus has been on the extinction chronology of individual or small groups of species, specific geographical regions or macroscale studies at very coarse geographical and taxonomic resolution, limiting the possibility of adequately testing the proposed hypotheses. We present, to our knowledge, the first global analysis of this extinction based on comprehensive country-level data on the geographical distribution of all large mammal species (more than or equal to 10 kg) that have gone globally or continentally extinct between the beginning of the Last Interglacial at 132,000 years BP and the late Holocene 1000 years BP, testing the relative roles played by glacial-interglacial climate change and humans. We show that the severity of extinction is strongly tied to hominin palaeobiogeography, with at most a weak, Eurasia-specific link to climate change. This first species-level macroscale analysis at relatively high geographical resolution provides strong support for modern humans as the primary driver of the worldwide megafauna losses during the late Quaternary.

Predator-prey interactions play an important role for species composition and community dynamics at local scales, but their importance in shaping large-scale gradients of species richness remains unexplored. Here, we use global range maps, structural equation models (SEM), and comprehensive databases of dietary preferences and body masses of all terrestrial, non-volant mammals worldwide, to test whether (1) prey bottom-up or predator top-down relationships are important drivers of broad-scale species richness gradients once the environment and human influence have been accounted for, (2) predator-prey richness associations vary among biogeographic regions, and (3) body size influences large-scale covariation between predators and prey. SEMs including only productivity, climate, and human factors explained a high proportion of variance in prey richness (R2 = 0.56) but considerably less in predator richness (R2 = 0.13). Adding predator-to-prey or prey-topredator paths strongly increased the explained variance in both cases (prey R2 = 0.79, predator R2 = 0.57), suggesting that predator-prey interactions play an important role in driving global diversity gradients. Prey bottom-up effects prevailed over productivity, climate, and human influence to explain predator richness, whereas productivity and climate were more important than predator top-down effects for explaining prey richness, although predator top-down effects were still significant. Global predator-prey associations were not reproduced in all regions, indicating that distinct paleoclimate and evolutionary histories (Africa and Australia) may alter species interactions across trophic levels. Stronger crosstrophic- level associations were recorded within categories of similar body size (e.g., large prey to large predators) than between them (e.g., large prey to small predators), suggesting that mass-related energetic and physiological constraints influence broad-scale richness links, especially for large-bodied mammals. Overall, our results support the idea that trophic interactions can be important drivers of large-scale species richness gradients in combination with environmental effects. © 2013 by the Ecological Society of America.

The development of eyespots on the wing surface of butterflies of the family Nympalidae is one of the most studied examples of biological pattern formation.However, little is known about the mechanism that determines the number and precise locations of eyespots on the wing. Eyespots develop around signaling centers, called foci, that are located equidistant from wing veins along the midline of a wing cell (an area bounded by veins). A fundamental question that remains unsolved is, why a certain wing cell develops an eyespot, while other wing cells do not. We illustrate that the key to understanding focus point selection may be in the venation system of the wing disc. Our main hypothesis is that changes in morphogen concentration along the proximal boundary veins of wing cells govern focus point selection. Based on previous studies, we focus on a spatially two-dimensional reaction-diffusion system model posed in the interior of each wing cell that describes the formation of focus points. Using finite element based numerical simulations, we demonstrate that variation in the proximal boundary condition is sufficient to robustly select whether an eyespot focus point forms in otherwise identical wing cells. We also illustrate that this behavior is robust to small perturbations in the parameters and geometry and moderate levels of noise. Hence, we suggest that an anterior-posterior pattern of morphogen concentration along the proximal vein may be the main determinant of the distribution of focus points on the wing surface. In order to complete our model, we propose a two stage reaction-diffusion system model, in which an one-dimensional surface reaction-diffusion system, posed on the proximal vein, generates the morphogen concentrations that act as non-homogeneous Dirichlet (i.e., fixed) boundary conditions for the two-dimensional reaction-diffusion model posed in the wing cells. The two-stage model appears capable of generating focus point distributions observed in nature.

We therefore conclude that changes in the proximal boundary conditions are sufficient to explain the empirically observed distribution of eyespot focus points on the entire wing surface. The model predicts, subject to experimental verification, that the source strength of the activator at the proximal boundary should be lower in wing cells in which focus points form than in those that lack focus points. The model suggests that the number and locations of eyespot foci on the wing disc could be largely controlled by two kinds of gradients along two different directions, that is, the first one is the gradient in spatially varying parameters such as the reaction rate along the anterior-posterior direction on the proximal boundary of the wing cells, and the second one is the gradient in source values of the activator along the veins in the proximal-distal direction of the wing cell.

We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3' untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors.

The AMPA-type glutamate receptor (AMPAR) subunit composition shapes synaptic transmission and varies throughout development and in response to different input patterns. Here, we show that chronic activity deprivation gives rise to synaptic AMPAR responses with enhanced fidelity. Extrasynaptic AMPARs exhibited changes in kinetics and pharmacology associated with splicing of the alternative flip/flop exons. AMPAR mRNA indeed exhibited reprogramming of the flip/flop exons for GluA1 and GluA2 subunits in response to activity, selectively in the CA1 subfield. However, the functional changes did not directly correlate with the mRNA expression profiles but result from altered assembly of GluA1/GluA2 subunit splice variants, uncovering an additional regulatory role for flip/flop splicing in excitatory signaling. Our results suggest that activity-dependent AMPAR remodeling underlies changes in short-term synaptic plasticity and provides a mechanism for neuronal homeostasis.

RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, facilitating fine tuning and homeostasis of cellular functions, a similar regulation has not been described for RNA editing. Here, we show that the AMPA receptor R/G editing site is dynamically regulated in the hippocampus in response to activity. These changes are bi-directional, reversible and correlate with levels of the editase Adar2. This regulation is observed in the CA1 hippocampal subfield but not in CA3 and is thus subfield/celltype-specific. Moreover, alternative splicing of the flip/flop cassette downstream of the R/G site is closely linked to the editing state, which is regulated by Ca(2+). Our data show that A-to-I RNA editing has the capacity to tune protein function in response to external stimuli.

PSD-95 is a prominent organizer of the postsynaptic density (PSD) that can present a filamentous orientation perpendicular to the plasma membrane. Interactions between PSD-95 and transmembrane proteins might be particularly sensitive to this orientation, as "long" cytoplasmic tails might be required to reach deeper PSD-95 domains. Extension/retraction of transmembrane protein C-tails offer a new way of regulating binding to PSD-95. Using stargazin as a model, we found that enhancing the apparent length of stargazin C-tail through phosphorylation or by an artificial linker was sufficient to potentiate binding to PSD-95, AMPAR anchoring, and synaptic transmission. A linear extension of stargazin C-tail facilitates binding to PSD-95 by preferentially engaging interaction with the farthest located PDZ domains regarding to the plasma membrane, which present a greater affinity for the stargazin PDZ-domain-binding motif. Our study reveals that the concerted orientation of the stargazin C-tail and PSD-95 is a major determinant of synaptic strength.

Unsustainable exploitation of wild animals is one of the greatest threats to biodiversity and to millions of people depending on wild meat for food and income. The international conservation and development community has committed to implementing plans for sustainable use of natural resources and has requested development of monitoring systems of bushmeat offtake and trade. Although offtake monitoring systems and indicators for marine species are more developed, information on harvesting terrestrial species is limited. Building on approaches developed to monitor exploitation of fisheries and population trends, we have proposed two novel indicators for harvested terrestrial species: the mean body mass indicator (MBMI) assessing whether hunters are relying increasingly on smaller species over time, as a measure of defaunation, by tracking body mass composition of harvested species within samples across various sites and dates; and the offtake pressure indicator (OPI) as a measure of harvesting pressure on groups of wild animals within a region by combining multiple time series of the number of harvested individuals across species. We applied these two indicators to recently compiled data for West and Central African mammals and birds. Our exploratory analyses show that the MBMI of harvested mammals decreased but that of birds rose between 1966/1975 and 2010. For both mammals and birds the OPI increased substantially during the observed time period. Given our results, time-series data and information collated from multiple sources are useful to investigate trends in body mass of hunted species and offtake volumes. In the absence of comprehensive monitoring systems, we suggest that the two indicators developed in our study are adequate proxies of wildlife offtake, which together with additional data can inform conservation policies and actions at regional and global scales.

There is increasing reliance on ecological models to improve our understanding of how ecological systems work, to project likely outcomes under alternative global change scenarios and to help develop robust management strategies. Two common types of spatiotemporally explicit ecological models are those focussed on biodiversity composition and those focussed on ecosystem function. These modelling disciplines are largely practiced separately, with separate literature, despite growing evidence that natural systems are shaped by the interaction of composition and function. Here we call for the development of new modelling approaches that integrate composition and function, accounting for the important interactions between these two dimensions, particularly under rapid global change. We examine existing modelling approaches that have begun to combine elements of composition and function, identifying their potential contribution to fully integrated modelling approaches. The development and application of integrated models of composition and function face a number of important challenges, including biological data limitations, system knowledge and computational constraints. We suggest a range of promising avenues that could help researchers overcome these challenges, including the use of virtual species, macroecological relationships and hybrid correlative-mechanistic modelling. Explicitly accounting for the interactions between composition and function within integrated modelling approaches has the potential to improve our understanding of ecological systems, provide more accurate predictions of their future states and transform their management.

The metabolism of luciferin in mice transgenic for luciferase (luc) produces light that may be detected trans vivo by an intensified CCD camera (biophotonics). Thus, the generation of transgenic promoter-luciferase animals for genes regulated by specific toxic processes, coupled with real-time evaluation of site-specific gene expression may provide novel, non-invasive biomarkers which are predictive of developing toxicity in vivo. As part of a programme to evaluate the potential of biophotonics for predictive toxicology we have conducted a series of studies in HO-1.luc transgenic mice. Male and female animals were treated with chloroform (200 mg/kg, p.o., daily for 5 days) and imaged 2 and 6 h after dosing. During a 2-day washout period, female animals were treated daily with testosterone prior to repeat administration of chloroform for a further 5 days. Comparison of the in vivo response of the luciferase reporter with markers of toxicity measured ex vivo (differential gene expression of adaptive antioxidant response genes, clinical chemistry and microscopic examination) confirms the gender-specific difference in chloroform renal toxicity in HO-1.luc transgenic mice and its reversal following androgenisation of females and correlates with the expression of the endogenous haem oxygenase-1 (HO-1) gene. These studies demonstrate the capacity of biophotonics for real-time site-specific gene expression, which may be predictive of developing toxicity.

Lemur tyrosine kinase 3 (LMTK3) is associated with cell proliferation and endocrine resistance in breast cancer. We found that, in cultured breast cancer cell lines, LMTK3 promotes the development of a metastatic phenotype by inducing the expression of genes encoding integrin subunits. Invasive behavior in various breast cancer cell lines positively correlated with the abundance of LMTK3. Overexpression of LMTK3 in a breast cancer cell line with low endogenous LMTK3 abundance promoted actin cytoskeleton remodeling, focal adhesion formation, and adhesion to collagen and fibronectin in culture. Using SILAC (stable isotope labeling by amino acids in cell culture) proteomic analysis, we found that LMTK3 increased the abundance of integrin subunits α5 and β1, encoded by ITGA5 and ITGB1. This effect depended on the CDC42 Rho family guanosine triphosphatase, which was in turn activated by the interaction between LMTK3 and growth factor receptor-bound protein 2 (GRB2), an adaptor protein that mediates receptor tyrosine kinase-induced activation of RAS and downstream signaling. Knockdown of GRB2 suppressed LMTK3-induced CDC42 activation, blocked ITGA5 and ITGB1 expression promoted by the transcription factor serum response factor (SRF), and reduced invasive activity. Furthermore, abundance of LMTK3 positively correlated with that of the integrin β1 subunit in breast cancer patient's tumors. Our findings suggest a role for LMTK3 in promoting integrin activity during breast cancer progression and metastasis.

In patients with breast cancer (BC), deregulation of estrogen receptor (ERα) activity may account for most resistance to endocrine therapies. Our previous study used a whole-human kinome siRNA screen to identify functional actors in ERα modulation and showed the implication of proteins kinase suppressors of ras (KSR1). From those findings we evaluated the clinical impact of KSR1 variants in patients with ERα+ BC treated with TAM. DNA was obtained from 222 patients with advanced ERα+ BC treated with TAM who had undergone surgery from 1981 to 2003. We selected three potentially functional relevant KSR1 polymorphisms; two within the 3'UTR (rs224190, rs1075952) and one in the coding exon 7 (rs2293180). The primary end points were overall survival (OS) and disease-free survival (DFS). After a 6.4-year median follow-up, patients carrying the rs2241906 TT genotype showed shorter DFS (2.1 vs 7.1 years, P=0.005) and OS (2.6 vs 8.4 years P=0.002) than those with the TC or TT genotypes. Those associations remained significant in the multivariable analysis adjusting age, lymph node status, LMTK3 and IGFR variants and HER2 status. The polymorphisms rs2241906 and rs1075952 were in linkage disequilibrium. No association was shown between rs2293180 and survival. Among the actors of ERα signaling, KSR1 rs2241906 variants may predict survival in patients with advanced ERα+ BC treated with adjuvant TAM.

Large-scale transcriptome and epigenome analyses have been widely utilized to discover gene alterations implicated in cancer development at the genetic level. However, mapping of signaling dynamics at the protein level is likely to be more insightful and needed to complement massive genomic data. Stable isotope labeling with amino acids in cell culture (SILAC)-based proteomic analysis represents one of the most promising comparative quantitative methods that has been extensively employed in proteomic research. This technology allows for global, robust and confident identification and quantification of signal perturbations important for the progress of human diseases, particularly malignancies. The present review summarizes the latest applications of in vitro and in vivo SILAC-based proteomics in identifying global proteome/phosphoproteome and genome-wide protein-protein interactions that contribute to oncogenesis, highlighting the recent advances in dissecting signaling dynamics in cancer.

Kinase suppressor of Ras 1 (KSR1) has been implicated in tumorigenesis in multiple cancers, including skin, pancreatic and lung carcinomas. However, our recent study revealed a role of KSR1 as a tumour suppressor in breast cancer, the expression of which is potentially correlated with chemotherapy response. Here, we aimed to further elucidate the KSR1-regulated signalling in response to genotoxic agents in breast cancer. Stable isotope labelling by amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS) was implemented to globally characterise cellular protein levels induced by KSR1 in the presence of doxorubicin or etoposide. The acquired proteomic signature was compared and GO-STRING analysis was subsequently performed to illustrate the activated functional signalling networks. Furthermore, the clinical associations of KSR1 with identified targets and their relevance in chemotherapy response were examined in breast cancer patients. We reveal a comprehensive repertoire of thousands of proteins identified in each dataset and compare the unique proteomic profiles as well as functional connections modulated by KSR1 after doxorubicin (Doxo-KSR1) or etoposide (Etop-KSR1) stimulus. From the up-regulated top hits, several proteins, including STAT1, ISG15 and TAP1 are also found to be positively associated with KSR1 expression in patient samples. Moreover, high KSR1 expression, as well as high abundance of these proteins, is correlated with better survival in breast cancer patients who underwent chemotherapy. In aggregate, our data exemplify a broad functional network conferred by KSR1 with genotoxic agents and highlight its implication in predicting chemotherapy response in breast cancer.

Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF-7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteomes, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes demonstrated distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signalling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer.

We documented one of the most species-rich assemblages of tropical rain-forest Auchenorrhyncha, comprising 402 phloem- and xylem-feeding species, by sampling adults from forest vegetation. Further, we reared 106 species from larvae sampled on 14 plant species. Both xylem- and phloem-feeding guilds exhibited wide host-plant ranges, as 74% of species fed on more than one plant family. In comparison, using data extracted from the temperate-zone literature, phloem-feeders exhibited lower host specificity in Papua New Guinea than in Germany, because in Papua New Guinea they were dominated by generalist Fulgoroidea while in Germany by specialist Membracoidea. The similarity of Auchenorrhyncha assemblages from different plant species was unrelated to the phylogenetic distance between their hosts. Host specificity, abundance and species composition of Auchenorrhyncha assemblages were unrelated to the optimum of their host plant species on succession gradient from secondary to primary forest. Higher host specificity did not lead to greater species richness in Auchenorrhyncha assemblages feeding on different plant species, but the number of species feeding on a particular plant species was a strong predictor of the Auchenorrhyncha abundance on that plant. These patterns suggest that Auchenorrhyncha assemblages on these plant species are not saturated with species and determined by division of limited resources among competitors, but instead are dependent on the number of colonizers from the regional species pool.

Reliable delimitation and identification of species is central not only to systematics, but also to studies of biodiversity, ecology and pest management. In the era of Internet-based biodiversity databases misidentifications are rapidly disseminated and may have far-reaching consequences. Leafhoppers from the genus Aphrodes (Hemiptera, Cicadellidae) are common and abundant, but, nevertheless, they are still a taxonomically challenging group whose members are often assessed in ecological studies and are also potential vectors of plant diseases. Previous study has shown that the syntype series for A. aestuarina (Edwards) includes also specimens of A. makarovi Zachvatkin and has suggested that misidentifications may be widespread in museum collections. We studied Aphrodes individuals collected from the U.K. and Slovenia in order to provide a more comprehensive analysis of this genus using multiple criteria. Combined work using male and female vibrational signals emitted during courtship, and a 600-bp fragment within the barcoding region of the COI mtDNA gene, provided validated specimens that we also used for morphometric study. Analyses confirmed A. aestuarina, A. bicincta, A. diminuta and A. makarovi as behaviourally, genetically and morphologically distinct species. Although any of these approaches could be used alone to distinguish between species, combining morphological and molecular approaches will help to improve reliability, especially when identifying females. Morphological investigation of validated individuals from the U.K. and Slovenia also revealed geographic differences within species. By combining several body and aedeagus morphological characters males can be reliably identified, however, morphological differences between species are, nevertheless, relatively small. By contrast, observed genetic distances between Aphrodes species are relatively large (4.2–7.0%). At about half of our collecting sites more than one Aphrodes species was found and A. makarovi was collected together with every other species, including A. aestuarina on tidal saltmarshes. Due to low morphological variation between syntopic congeners it is likely that many museum specimens of Aphrodes have been assigned to the wrong species and species identification in ecological and vector studies may also be questionable.

In sexual communication, partners often form a duet, an exchange of species- and sex-specific signals, and in such systems mate recognition is likely to be reciprocal. We studied the role of vibrational signals in reproductive isolation in the genus Aphrodes (Hemiptera: Cicadellidae) in which mate recognition is based on highly divergent male advertisement calls and similar female replies. We first determined in playback experiments the preferences of females of four Aphrodes species to conspecific and heterospecific male advertisement calls as well as to species-specific elements in these calls. Females of all four species responded preferentially to calls of conspecific males; however, male calls composed of similar elements played only a limited role in mate recognition. In particular, females of Aphrodes aestuarina and Aphrodes bicincta showed higher responsiveness to each other's male calls than to calls of other species. In this species pair we further examined the role of female signals and duet structure in assortative mating using ‘no-choice’ mating experiments. The generally higher responsiveness of A. aestuarina females to male calls of A. bicincta did not translate into higher mating success in this heterospecific cross; lengthy replies of A. aestuarina females resulted in a breakdown of a complex species-specific duet structure and associated difficulties in locating the female reduced the probability of heterospecific mating. Our study shows that in mating systems based on a duet, males may contribute more than females to sexual isolation between species. Males' contribution to assortative mating may stem not only from mate recognition but also from inability to locate the source of the heterospecific female reply.

Species reintroduction programmes should include consideration of potential impacts on key species in the recipient community. Wild boar (Sus scrofa) have been reintroduced into Britain after a 700-year absence. There is an urgent need to understand how this ecosystem engineer will affect plant communities in the habitats that it invades. We investigated the impact of rooting by wild boar on bluebells (Hyacinthoides non-scripta), a species that is highly valued for its impressive floral displays and is an important and legally protected component of the UK forest flora. We monitored bluebell performance over three growing seasons in woodland habitats that are routinely rooted by boar in southern England. H. non-scripta cover and reproductive performance were monitored in small-scale experimental exclosures to exclude boar, compared to open control plots, set up on areas that either had or had not been previously rooted. Immediate effects were that rooting significantly reduced the percentage cover and density of H. non-scripta plants, by up to 95 and 60 %, respectively, and also adversely affected the number of flowering stems. However, there was evidence that cessation of rooting brought about by excluding the boar enabled substantial recovery in percentage cover and the density of flowering stems within 2 years. A positive effect of rooting on germination may have assisted this recovery. Thus, the impact of wild boar rooting on bluebell populations is locally severe, but there is potential for rapid recovery if plants are protected. Long-term effects of sustained or frequently repeated rooting still need to be investigated.

Invasive alien species (IAS) are considered one of the greatest threats to biodiversity, particularly through their interactions with other drivers of change. Horizon scanning, the systematic examination of future potential threats and opportunities, leading to prioritization of IAS threats is seen as an essential component of IAS management. Our aim was to consider IAS that were likely to impact on native biodiversity but were not yet established in the wild in Great Britain. To achieve this, we developed an approach which coupled consensus methods (which have previously been used for collaboratively identifying priorities in other contexts) with rapid risk assessment. The process involved two distinct phases:

1. Preliminary consultation with experts within five groups (plants, terrestrial invertebrates, freshwater invertebrates, vertebrates and marine species) to derive ranked lists of potential IAS.
2. Consensus-building across expert groups to compile and rank the entire list of potential IAS.

Five hundred and ninety-one species not native to Great Britain were considered. Ninety-three of these species were agreed to constitute at least a medium risk (based on score and consensus) with respect to them arriving, establishing and posing a threat to native biodiversity. The quagga mussel, Dreissena rostriformis bugensis, received maximum scores for risk of arrival, establishment and impact; following discussions the unanimous consensus was to rank it in the top position. A further 29 species were considered to constitute a high risk and were grouped according to their ranked risk. The remaining 63 species were considered as medium risk, and included in an unranked long list. The information collated through this novel extension of the consensus method for horizon scanning provides evidence for underpinning and prioritizing management both for the species and, perhaps more importantly, their pathways of arrival. Although our study focused on Great Britain, we suggest that the methods adopted are applicable globally.

1. The exploitation of shared resources by diverse organisms underpins the structure of ecological communities. Hemiparasitic plants and the insect herbivores feeding on them both rely, directly and indirectly, on the resources supplied by the parasite's host plant. Therefore, the identity and number of host plant species providing these resources is likely to be critical for parasite and herbivore performance.

2. We tested the effect of single and multiple host species on the biomass of the generalist parasitic plant Rhinanthus minor and the abundance of its aphid (Aphis gossypii) herbivores.

3. Parasite biomass was proportional to the number of haustorial connections to host roots and was determined by host species identity rather than host functional group. Host species identity was also an important influence on aphid population size, and parasites attached to Lotus corniculatus experienced a considerable reduction in aphid herbivory.

4. The effects on the parasite attaching to multiple hosts depended on the combination of species present. However, host mixtures generally benefitted aphids by diluting the negative effects of particular host species.

5. Our findings suggest that the specificity of host attachment alters the impact of this keystone parasitic plant on its own herbivores and, potentially, on the wider plant and herbivore community.

This study investigated cerambycid long-horned beetles in a lowland tropical forest in Fiji and produced 18 new records of beetle-tree associations along with data on beetle phenology and development times. Beetles were reared from timber baits exposed for 1 month to ovipositing females in the Savura Forest Park, Viti Levu. Twelve native, locally common tree species representing 10 families were examined. For each tree, two baits consisting of 16 kg of freshly cut branches were exposed in each of four time periods between June 2008 and May 2009. Eighteen cerambycid species and 557 individual beetles were reared from the 96 baits, with three of the beetle species probably being undescribed. Ceresium was the most abundant genus, representing almost 90% of all individuals reared, with most adults emerging between 4 and 6 months after the timber baits were exposed. Seventeen of the 18 beetle species each emerged from timber belonging to a single tree species, although more rearing records are required to support the high level of host-plant specificity reported here.

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.

LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.

Sodium 2-mercaptoethanesulphonate (MESNA) is a uroprotective agent generally given i.v. to prevent haemorrhagic cystitis during oxazaphosphorine cancer chemotherapy. Oral administration of the drug is described since this might be an important route during long-term oxazaphosphorine treatment. MESNA is absorbed from the GI tract and excreted in the urine (about 41.5% of the dose), peak excretion being 2-3 hr after administration. A proportion of the excreted dose is as free thiols (about 24.2%) and the remainder is as disulphides. MESNA is shown to enhance excretion of cysteine in urine.

The effects of the homologous series of carboxylic esters, methyl propionate to methyl decanoate, on the steady-state inactivation of the sodium current in squid axons have been studied. The esters moved the relationship between the inactivation parameter, h infinity, and the membrane potential in the hyperpolarizing direction, thus reducing the number of sodium channels available at the resting potential. The concentration dependence of the shift at the mid-point of the curve of h infinity against potential has been measured for all esters except decanoate, which was almost inactive. Two aspects of these concentration dependences suggest that molecular volume is an important determinant of the effectiveness of each ester. Firstly, there is a sharp decline in activity above methyl hexanoate. This cut-off in activity resembles that for hydrocarbons where it has been suggested [e.g., Haydon, D.A., Urban, B.W. 1983, J. Physiol. (London) 341:411-427] to a result from a decrease in uptake with increasing molecular volume. (Further data for the hydrocarbons n-butane to n-heptane are reported here.) Secondly, the smallest compounds, methyl propionate and methyl butyrate, are less effective than would be predicted if equal membrane concentrations of each ester produced the same shift. The aqueous concentration dependences for these esters indicate that below methyl hexanoate, as the series is descended, progressively higher membrane concentrations are required to produce a given shift. This would be expected if the volume of ester in the membrane, rather than the number of molecules, is important.(ABSTRACT TRUNCATED AT 250 WORDS)

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.

The effects of the n-alcohols from pentanol to dodecanol on nAChR channel function were resolved at the single channel level. ACh-activated channel activity was recorded from isolated membrane patches using the patch clamp method. The intermediate-chain alcohols (C5-C8) had two main effects: (1) They caused channel openings to be interrupted by brief shut or blocked periods, the duration of which was dependent on chain length of the alcohol but independent of concentration. (2) They caused a reduction in the duration of bursts of openings. The long-chain alcohols (C9-C11) produced only the second effect, and there was a decline in activity beyond undecanol. Results were consistent with a mechanism of channel blockade and were analyzed in terms of an open channel block model with a long-lived closed-blocked state beyond the blocked state. Affinity for the binding site increased with chain length up to octanol. The standard free energy per methylene group for adsorption to the site was calculated to be -3.3 kJ/mol, indicating the very hydrophobic nature of the site.

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-D-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7(-/-) mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7(-/-) strains has important implications for our understanding of the role of this receptor in health and disease.

Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. All except P2X6 assemble as homotrimers, and six heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) have been described. In addition, P2X4 homomers associate with P2X2 or P2X7 homomers as dimers of trimers. The various P2X receptors show individual functional properties, suggesting distinct physiological roles. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits interact.

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na(+) and N-methyl-D-glucamine (NMDG(+)) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.

Voltage-gated K(+) channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

Depletion of Ca(2+) from the endoplasmic reticulum (ER) lumen triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca(2+) store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.

P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells.

The functional expression of P2X receptors at the plasma membrane is dependent on their trafficking along secretory and endocytic pathways. There are seven P2X receptor subunits, and these differ in their subcellular distributions because they have very different trafficking properties. Some are retained within the endoplasmic reticulum (ER), while others are predominantly at the cell surface or within endosomes and lysosomes. Changes in recruitment of receptors to and from the plasma membrane provides a way of rapidly up- or down-regulating the cellular response to adenosine triphosphate (ATP). An additional layer of regulation is the targeting of these receptors within the membranes of each compartment, which affects their stability, function and the nature of the effector proteins with which they form signaling complexes. The trafficking and targeting of P2X receptors is regulated by their interactions with other proteins and with lipids and we can expect this to vary in a cell-type specific manner and in response to changes in the environment giving rise to differences in receptor activity and function.

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7(-/-) mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an approximately 4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.

P2X receptors are cation selective ion channels gated by the binding of extracellular ATP. Seven subtypes have been identified and they have widespread and overlapping distributions throughout the body. They form homo- and heterotrimeric complexes that differ in their functional properties and subcellular localization. They form part of larger signalling complexes, interacting with unrelated ion channels and other membrane and cytosolic proteins. Up- or down-regulation of their expression is associated with several disease states. This review aims to summarize recent work on the assembly and trafficking of this family of receptors.

P2X4 and P2X7 are the predominant purinergic receptor subtypes expressed in macrophages, microglia and epithelial cells, and they are potentially important therapeutic targets for treatment of pain and inflammation. For both subtypes, there is evidence that plasmamembrane expression is tightly regulated. P2X4 receptors are prominently localized to lysosomes and resist degradation by virtue of N-linked glycans decorating the intra-luminal loop of the receptor. P2X7 receptors are reported to be predominantly intracellular in monocytes and are upregulated at the plasma membrane upon differentiation of monocytes to macrophages. We have previously shown an interaction between P2X4 and P2X7 receptors, suggesting that they might form an association. The mechanisms that regulate their plasma membrane expression are not well understood, and we have used biochemical methods to look at the size and distribution of the native complexes in a variety of cell types in which they are co-expressed. We have compared the proportion of receptors expressed at the cell surface in cultured microglia and macrophages following exposure to modulators of microglial/macrophage activation. Surface expression was analysed by biotinylation of exposed proteins and by cross-linking proteins with membrane impermeant cross-linkers, followed by SDS-PAGE and western blotting. The modulators included lipopolysaccharide (LPS), ATP and phorbol esters. Cross-linking of surface receptors also provides a means of analysing the subunit composition of the complexes at the plasma membrane, based upon the size difference of P2X4 and P2X7 subunits. These results are compared with those obtained using blue native (BN)-PAGE analysis of the total P2X receptor population.

Activation of P2X7 receptors triggers an increase in membrane permeability to large molecules and, in immune cells, the processing and release of inflammatory cytokines. The hemi-channel Pannexin-1 (Px1) was recently shown to interact with P2X7 and to be involved in both processes [1]. There is also evidence that the P2X4 receptor interacts with P2X7 [2]. In this study, we set out to further investigate the trafficking and functional interaction between P2X7, Px1 and P2X4. Proteins were overexpressed in HEK293 cells, and we used siRNA and carbenoxolone (CBX) to inhibit Px1. In HEK293 cells transiently overexpressing P2X7, incubation with CBX produced a dose-dependent inhibition of ethidium uptake evoked by ATP or BzATP. Surprisingly, in HEK293 cells expressing recombinant Px1 as well as P2X7, there was a reduction in the rate of ethidium uptake compared to cells transfected with P2X7 alone, and also incubation with CBX enhanced rather than inhibited BzATP-evoked ethidium uptake. The expression levels of P2X7 receptors in wild-type and Px1 stable cells were very similar, yet the fraction of P2X7 at the cell surface was slightly increased in Px1-cells as determined by biotinylation and selective cross-linking of surface receptors. The role of the cytoskeleton in large pore formation and in P2X7 coupling to Px1 was also examined. As shown previously, disruption of actin filaments with latrunculin had no effect on ethidium uptake; in contrast, depolymerisation of microtubules with colchicine resulted in a significant increase in both the rate and maximal dye uptake in response to ATP. The effects of co-expressing P2X4 on the interaction between P2X7 and Px1 are being investigated.

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7–/– mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ∼4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.

BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7 -/- mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function. © 2011 The British Pharmacological Society.

The rodent P2X7k splice variant, which utilizes a different exon 1 and is expressed in both wild type and the Glaxo P2X7−/−mice, has at least a 10-fold greater sensitivity to agonists ATP and BzATP than the full length mouse P2X7a variant, as determined from patch clamp experiments. ThemP2X7k receptor also coupled to the rapid uptake of the cationic dye ethidium, and this process was unaffected by the SNP, P451L, that previously was shown to inhibit mP2X7a receptor-mediated dye uptake. In HEK293 cells, activation of mP2X7k also triggered the uptake of the anionic dye Lucifer yellow, the rate of which was dramatically increased by a 5-min pre-incubation with the pannexin-1 inhibitor, carbenoxolone (CBX) at 37°C. CBX also increased mP2X7k-mediated ethidium uptake but the Ca2+ signal evoked by activation of mP2X7k was unaffected, suggesting that CBX selectively affects ‘large pore’ formation. Themechanismof action of CBX is unclear but the results are not consistentwith pannexin-1 being the dye uptake pathway. P2X7k is expressed in mice spleen T-lymphocytes where NAD is reported to be a physiologically important agonist. In HEK293 cells, 10 uM NAD stimulated mP2X7k-mediated ethidium uptake whereas mP2X7a did not respond to concentrations up to 300 uM NAD. NAD also stimulated a rapid Ca2+ response in HEK293 cells expressing mP2X7k. The coexpression of mP2X7a and mP2X7k in HEK cells reduced the amplitude of the NAD response compared to cells expressing mP2X7k alone. Our results suggest the expression of P2X7k increases the diversity of P2X7 receptor signaling in rodents.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons.

The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7.

P2X4 receptors have a widespread distribution throughout the body. In primary cells where we have examined their subcellular distribution, including macrophages, microglia and endothelial cells, the receptors are localized to late endosomes and lysosomes (LEL). Mutational analysis of heterologously expressed receptors has shown that a C-terminal tyrosine based motif and N-terminal dileucine-like motif are both involved in targeting the receptor to LELs. Within lysosomes, P2X4 receptors resist degradation by virtue of N-linked glycans, and they can be delivered from lysosomes to the surface to increase the expression of functional receptors. In activated microglia in culture, a proportion of receptors traffic to and from the cell surface in a dynamic manner and further activation of microglia by LPS was shown to increase the number at the cell surface. In cultured bone marrow derived macrophages, however, receptors were localized exclusively to LELs and no expression was detected at the surface unless lysosome exocytosis was stimulated by incubation with a weak base. We are interested in whether or not P2X4 receptors can function within the acidic environment of the lysosome or phagosome, as well as at the plasma membrane. Intracellular P2X-like receptors have been identified in Dictyostelium and are thought to function within the acidic environment of the contractile vacuole. With the extracellular solution at lysosomal pH (4.6), P2X4 receptors at the cell surface of HEK293 cells can be activated by millimolar extracellular ATP. We are examining the effects of wild type and mutant P2X4 receptor expression on LEL function and calcium signalling.

Contributing to the diversity of human and rodent P2X7 receptors are alternatively spliced variants. Here, we describe two murine splice variants with C-terminal truncations (ΔC) that arise from the use of alternative exon 13 s. P2X7(13b) terminates at residue 430, whereas P2X7(13c) possesses an additional 11-amino acid stretch after residue 430. The tissue distribution and structural and functional characteristics of the ΔC variants were investigated and compared to the fulllength variant, P2X7(a). RT-PCR analysis using tissue from the wild-type C57BL strain of mice indicated that the ΔC variants are expressed in a range of tissues including brain, salivary gland and spleen. The recombinant receptors expressed in HEK293 cells were analysed by BN-PAGE and cross-linking studies and both ΔC splice variants formed stable homotrimers. Whole-cell patch-clamp recordings revealed that BzATP-induced currents were at least an order of magnitude smaller for the ΔC variants compared to P2X7(a) and the EC50 value was 1.3 mM. Co-expression of P2X7(13b) with P2X7 (a) reduced the amplitude of 1 mM BzATP-evoked currents by ∼3-fold compared to P2X7(a) alone and shifted the EC50 value from 320 μM to 410 uM, suggesting that the ΔC variant exerts a dominant negative effect. Expression of P2X7 receptor complexes in tissue from wild type and Pfizer P2X7−/− mice was analyzed by BN-PAGE and using cross-linkers. Blots using an antibody to the extracellular domain of P2X7 showed trimers of a size consistent with the ΔC variants in spleen and salivary gland from Pfizer P2X7−/− mice, suggesting that these mice are not null for P2X7 expression.

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth.

1. The actions of the n-alcohols from pentanol to dodecanol on nicotinic acetylcholine receptor (nAChR) channels were investigated by recording single ACh-activated channel activity from inside-out membrane patches isolated from cultured rat myotubes. Alcohols were applied to the cytoplasmic side of the membrane; aqueous concentrations ranged from 11.7 mM-pentanol to 0.02 mM-dodecanol. 2. The intermediate-chain alcohols (pentanol to octanol) caused channel currents to fluctuate between the fully open and closed state level so that openings occurred in bursts interrupted by brief gaps. Closed time distributions were fitted well with two exponential components, the fast component representing the closures within a burst. The number of gaps within a burst was dependent on alcohol concentration whereas gap duration was independent of concentration but increased with increasing chain length of the alcohol up to octanol. 3. Nonanol and decanol reduced the mean duration of bursts of openings but did not cause an increase in the number of short closed intervals within a burst. Beyond decanol there was a decline in the ability of the n-alcohols to affect channel function. A saturated solution of undecanol (0.07 mM) reduced the mean open time by 33 +/- 17%, whereas a saturated solution of dodecanol had no significant effect. 4. The current integral per burst was reduced by all the n-alcohols between pentanol and undecanol. The IC50S were as follows: hexanol, 0.53 +/- 0.14 mM; heptanol, 0.097 +/- 0.02 mM; octanol, 0.04 mM and nonanol, 0.16 +/- 0.035 mM. 5. The results were analysed in terms of an open channel block model with a long-lived closed-blocked state beyond the blocked state. Over the range of concentrations tested this describes the effects of all the n-alcohols (C5 to C12) on channel gating reasonably well. 6. Blocking rate constants (k+B) for pentanol through to nonanol were calculated to be between 2.8 and 5.7 X 10(6) M-1 S-1. These values are based on the assumption that the concentration of the alcohols at their site(s) of action was equal to the aqueous concentration applied to the membrane. 7. Equilibrium dissociation constants (KD), calculated from the blocking and unblocking rate constants (KD = k-B/k+B), decreased with increasing chain length from 8 mM for pentanol to 0.15 mM for octanol. The standard free energy per methylene group for adsorption to the site of action was calculated to be about -3.3 kJ mol-1.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X(2) receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm(3)) also points to a trimeric structure. P2X(2) receptors bearing His(6) epitope tags were incubated with anti-His(6) antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123 degrees , again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X(6) receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm(3). We conclude that P2X(2) receptors are trimers, whereas the P2X(6) receptor subunits do not form stable oligomers.

During clathrin-mediated endocytosis, proteins on the cell surface are selected for inclusion in clathrin-coated vesicles by clathrin adaptors, mainly the adaptor complex AP2. The P2X4 subtype of ATP-gated ion channel has in its C-terminus two putative endocytic motifs: a canonical YXXPhi motif and a non-canonical YXXGPhi motif (YEQGL). We demonstrate that endocytosis of P2X4 receptors is mediated preferentially by the YXXGPhi motif because the YXXPhi motif is inaccessible to AP2 owing to the structure of the channel. The crystal structure of a complex between residues 160-435 of the mu2 subunit of AP2 and a P2X4 C-terminal peptide showed that the YEQGL motif binds to mu2 at the same site as YXXPhi motifs. Y and Phi residues are accommodated in the same hydrophobic pockets in mu2 with the extra residue between them being accommodated by changes in the peptide's backbone configuration, when compared to YXXPhi motifs. These data demonstrate that the family of potential tyrosine-based endocytic signals must be expanded to include motifs with an additional glycine at Y+3 (YXXGPhi).

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling.

The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors.

The P2X(4) receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X(4) receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X(4) receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X(4) receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca(2+) ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X(4) receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X(4) receptor currents across the plasma membrane. Taken together, our data suggest that the P2X(4) receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.

ATP-gated P2X receptors are trimeric complexes formed by the homomeric or heteromeric assembly of seven different subunits. We have shown previously that, unlike all of the other P2X subunits, the P2X6 subunit cannot form homomeric receptors and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (J Biol Chem 280: 107591-10765, 2005). However, other studies have shown that P2X6 can form functional heteromeric receptors with P2X2 and P2X4 subunits. In this study, we used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy to investigate the assembly and trafficking of the P2X6 subunit, both alone and as part of a heteromer. We show that as a heteromer, it exits the ER and is either stably expressed at the cell surface or constitutively internalized, depending on its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N terminus of P2X6 exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X6 forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X2 receptor. The N-terminal mutants were, however, nonfunctional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X2 increased ER retention but was not sufficient to prevent the formation of functional homomeric receptors. We propose that the N terminus of the P2X6 subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of nonfunctional P2X receptors.

Cells can change their function by rapidly modulating the levels of certain proteins at the plasma membrane. This rapid modulation is achieved by using a specialised trafficking process called constitutive cycling. The constitutive cycling of a variety of transmembrane proteins such as receptors, channels and transporters has recently been directly demonstrated in a wide range of cell types. This regulation is thought to underlie important biological phenomena such as learning and memory, gastric acid secretion and water and blood glucose homeostasis. This review discusses the molecular mechanisms of constitutive cycling, its regulation by extracellular agents such as hormones and its misregulation in disease states.

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X(4) receptor. Three amino acids in the C terminus of P2X(4) (Tyr(378), Gly(381), and Leu(382)) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X(4) protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X(4) C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the mu2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X(4) receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X(4) receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.

G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gbetagamma subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gbetagamma but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2 binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.

1. The ATP-activated P2X2(a) and P2X2(b) receptor splice variants, which differ only in their C-terminal sequences, desensitize at different rates. We used mutational analysis to investigate the involvement of the C-terminal region in receptor desensitization. Rat wild-type and mutant P2X2 receptors were expressed in Xenopus oocytes and currents were measured using the two-electrode voltage-clamp technique. 2. Truncating P2X2 at the Lys369 splice site increased the rate of desensitization by >100-fold. Recovery from desensitization was slowed by approximately 5-fold. 3. Addition of Val370 onto the C-terminus of the truncated receptor slowed desensitization by approximately 70-fold. Point mutations that substituted either smaller or larger hydrophobic amino acids for Val370, within the P2X2(a) splice variant, had profound effects on the rate of desensitization. The rate decreased with increasing hydrophobicity but was not dependent upon the precise structure of the side group. 4. A mutant receptor, with only nine amino acids, Val-Asp-Pro-Lys-Gly-Leu-Ala-Gln-Leu, beyond the Lys369 splice site, desensitized at a similar rate to P2X2(a). Injection of the peptide of this sequence into oocytes expressing P2X2(a) increased the rate of desensitization, whereas the eight-residue peptide lacking the valine had no effect. 5. Neutralizing lysines in the vicinity of the splice site increased the rate of receptor desensitization. Substituting glutamine for Lys365 produced the greatest effect ( approximately 30-fold increase), whereas mutating lysines that were further upstream or downstream of this position had progressively less of an effect. 6. We conclude that the C-terminal splice site of the P2X2 receptor is located within a region that is critically involved in regulating the rate of receptor desensitization. The valine at position 370 interacts with an intracellular hydrophobic site to slow the rate of desensitization. Nearby lysines may facilitate this interaction.

1. G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gbetagamma and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1-GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1-GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4, 5-bisphosphate (PIP2). 2. The rate of inhibition of GIRK1-GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3. The dose-response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.

P2X receptors within the CNS mediate excitatory synaptic transmission and also act presynaptically to modulate neurotransmitter release. We have studied the targeting and trafficking of P2X4 and P2X2 receptors heterologously expressed in cultured olfactory bulb neurons. Homomeric P2X4 receptors had a punctate distribution, and many of the puncta colocalized with early endosomes. In contrast, P2X2 receptors were primarily localized at the plasma membrane. By antibody-labeling of surface receptors in living neurons, we showed that P2X4 receptors undergo rapid constitutive internalization and subsequent reinsertion into the plasma membrane, whereas P2X2 receptors were not regulated in such a way. The internalization of P2X4 receptors was dynamin-dependent, and the binding of ATP enhanced the basal rate of retrieval in a Ca2+-independent manner. The presence of the P2X4 subunit in a P2X4/6 heteromer governed the trafficking properties of the receptor. P2X receptors acted presynaptically to enhance the release of glutamate, suggesting that the regulated cycling of P2X4-containing receptors might provide a mechanism for modulation of synaptic transmission.

The cognitive enhancer XE991 interacts with K(+) channels consisting of KCNQ2 and KCNQ3 heteromultimers to block the M-current. XE991 can also block KCNQ1 K(+) channels expressed in oocytes, but sensitivity is reduced when the channels are coexpressed with minK (KCNE1). The purpose of the study was to examine the interaction of XE991 with other types of K(+) channel, especially those in the basolateral membranes of murine epithelia. K(+) channel blockade was measured by the inhibition of chloride secretion resulting from depolarization. XE991 inhibited the chloride secretory current in colonic epithelia by an interaction with basolateral K(+) channels when forskolin was used as the stimulus. However, when 1-ethyl-2-benzimidazolinone (EBIO) was used to stimulate chloride secretion, XE991 was ineffective unless charybdotoxin was also present. Because EBIO also activates Ca(2+)-sensitive K(+) channels, whereas forskolin activates only cAMP-sensitive K(+) channels, it is concluded that the latter are the targets for XE991. XE991 had effects similar to those of 293B on epithelial chloride transport, for which the target is known to be KCNQ1/KCNE3 multimers. mRNA for both these components of the cAMP-sensitive K(+) channels were found in high abundance in the colon, whereas KCNE1 was barely detectable. Furthermore, both XE991 and 293B were active in colonic epithelia from KCNE1 knockout mice. By contrast, in nasal epithelium, the forskolin sensitive chloride secretory current was barely sensitive to XE991 but was sensitive to clofilium. Xenopus laevis oocytes in which both KCNQ1 and KCNE3 had been expressed were significantly more sensitive to XE991 than oocytes expressing only KCNQ1.

cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2 subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2 subunit were injected intoXenopus laevis oocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b) receptor were not significantly different from those at the P2X2(a)homomeric channel. However, the P2X2(b) receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a) receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b)receptor. When the P2X2(a) and P2X2(b) receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b)subunit. In situ hybridization of the three newly isolated isoforms of the P2X2 subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b) and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d) splice variant was present only in the nodose ganglion, at a low level.

Voltage-gated K+ channels activate with depolarization of the membrane potential and subsequently inactivate. Shaker K+ channels inactivate very rapidly with a time constant of 2-3 ms. Aldrich and co-workers showed that this rapid inactivation arises from an amino(N)-terminal inactivation domain acting as a tethered particle that blocks the open channel. This mechanism is analogous to the “ball-and-chain” model proposed by Bezanilla and Armstrong for inactivation of Na+ channels. Removing the cytoplasmic N-terminal domain of Shaker K+ channels disrupts rapid inactivation, and internal application of a synthetic peptide corresponding to this domain (residues 1-20) restores inactivation. Several pieces of evidence indicate that block of Shaker channels by the inactivation domain and synthetic (ShB) peptide involves a single blocking particle physically occluding the pore. Recovery from inactivation and peptide block are speeded by raising the concentration of K+ on the opposite (external) side of the channel. Although the inactivation domain of the Shaker channel has a net positive charge, N-type inactivation shows no apparent voltage sensitivity over the range of -30 to +50 mV. 9 Peptide binding does show some voltage dependence, but increasing the net charge on the peptide does not increase this voltage dependence. Thus, the majority of the blocking particle does not appear to enter into the membrane electric field, but binds instead at the mouth of the pore.

1. The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein-gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild-type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2. Co-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1-Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine-induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4. Individually substituting the N- and C-terminal tails of Kir3.1 for those of Kir3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher ratio of ACh-induced to basal activity than the Kir3.1-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substituting the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core and C-terminal regions of Kir3.1 independently conferred time dependence on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.

We have examined how NGF-dependent rat sympathetic neurons maintain Ca2+ homeostasis when challenged with high K+ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K+ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca2+ channels within 2 days. Although resting [Ca2+]i was elevated by 1.6-fold, this was 50% less than the rise in [Ca2+]i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca2+]i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca2+ current, suggesting that voltage-sensitive Ca2+ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K+ medium with no significant rise in resting [Ca2+]i, although neurites did not grow. Neither Ca2+ channel density nor resting [Ca2+]i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca2+. However, the dihydropyridine antagonist nitrendipine blocked both high K(+)- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca2+]i above resting levels to survive with either high K+ or CPTcAMP, but dihydropyridine-sensitive Ca2+ channel activity may be essential for their survival promoting actions.

Kir3.1 and Kir3.2 associate to form G-protein-activated, inwardly rectifying K+ channels. To identify regions involved in the coassembly of these subunits, truncated Kir3.1 polypeptides were coexpressed with epitope-tagged subunits in an in vitro translation system. N-terminal, C-terminal, and core region polypeptides were coimmunoprecipitated with both Kir3.2 and Kir3.1, suggesting that multiple elements distributed throughout the Kir3.1 polypeptide contribute to intersubunit binding interactions. The Kir3.2 C-terminal polypeptide coimmunoprecipitated with the Kir3.1 C-terminal polypeptide, but neither region recognized the N-terminal domain and core region of the Kir3.1 subunit. This suggests that within Kir3 channels the C-terminal domains of neighboring subunits interact. Coexpression of the truncated polypeptides with Kir3.1 and Kir3.2 in Xenopus oocytes reduced functional expression of the heteromeric channels. Constructs encoding the core region plus N-terminal and proximal C-terminal regions competed more effectively than the core region alone, which supports the contribution of all three regions to intersubunit binding interactions. Proximal and distal segments of the C-terminal domain were as effective at inhibiting functional expression as the entire C-terminal domain.

Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage-dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.

Waste management is important in insect societies because waste can be hazardous to adults, brood and food stores. The general organization of waste management and the influence of task partitioning, division of labor and age polyethism on waste processing were studied in three colonies of the tropical American stingless bee Melipona beecheii Bennett in Yucatán, Mexico. Waste generated in the colony (feces, old brood cells, cocoons, dead adults and brood) was collected by workers throughout the nest and taken to specific waste dumps within the nest. During the day, workers based at the waste dumps formed waste pellets, which they directly transferred in 93% of cases, to other workers who subsequently removed them from the nest. This is an example of task partitioning and is hypothesized to improve nest hygiene as has been found in leafcutting ants, Atta. To investigate division of labor and age polyethism we marked a cohort of 144 emerging workers. Workers forming waste pellets were on average 31.2±6.5 days old (±SD, N= 40, range of 18-45 days). The life span of M. beecheii workers was 49.0±14.0 days (N= 144). There was no difference in the life span of workers who formed (52.2±11.6 days, N= 40) or did not form (49.9±11.5 days, N= 97) waste pellets, suggesting that waste work did not increase mortality. Although waste was probably not hazardous to adults and brood, because the dumps are located outside the brood chamber, its presence inside the nests can attract phorid flies and predators, which can harm the colony.

Inclusive fitness benefits depend on recognizing the right individuals to interact with. Social insect nests protect themselves from non-kin intruders through nestmate recognition based on chemical cues. The recognition cues on adult individuals are from a mixture of genetic and environmental sources, but the ontogeny and use of recognition cues on eggs has not been previously assessed. We studied recognition by workers of eggs that were either nestmates or non-nestmates. and the ontogeny of recognition cues on eggs in the ant Formica fusca, a species with precise egg recognition abilities. Workers were able to discriminate among freshly laid eggs with no nest derived cues on them, and the egg surface chemicals varied among nests in these eggs, suggesting that queen derived cues are used in nestmate recognition. The results are discussed in the light of their implications on deceptive social parasite strategies and within colony conflicts.

Worker honeybees, Apis mellifera, police each other’s reproduction by killing worker-laid eggs. Previous experiments demonstrated that worker policing is effective, killing most (∼98%) worker-laid eggs. However, many queen-laid eggs were also killed (∼50%) suggesting that effective policing may have high costs. In these previous experiments, eggs were transferred using forceps into test cells, mostly into unrelated discriminator colonies. We measured both the survival of unmanipulated queen-laid eggs and the proportion of removal errors that were rectified by the queen laying a new egg. Across 2 days of the 3-day egg stage, only 9.6% of the queen-laid eggs in drone cells and 4.1% in worker cells were removed in error. When queen-laid eggs were removed from cells, 85% from drone cells and 61% from worker cells were replaced within 3 days. Worker policing in the honeybee has a high benefit to policing workers because workers are more related to the queen’s sons (brothers, r = 0.25) than sister workers’ sons (0.15). This study shows that worker policing also has a low cost in terms of the killing of queen-laid eggs, as only a small proportion of queen-laid eggs are killed, most of which are rapidly replaced.

Urbanization is increasing worldwide. Urban habitats often support considerable biodiversity and so are of conservation value, even though they are highly modified ecosystems. Urban parks and gardens are rich in flowers that provide food for pollinators, including bees. Here, we use waggle dance decoding to investigate foraging by 3 honey bee hives located in the city of Brighton, UK, over almost an entire foraging season, April to October. Waggle dances were recorded using video cameras and decoded during framewise playback on a computer by measuring the angle and duration of the waggle phase. Foraging was mostly local (mean monthly distances 0.5–1.2 km) and mostly within the surrounding urban area (monthly means 78–92 %) versus the countryside (closest distance 2.2 km) even though this was well within the honey bee maximum foraging range (c. 12 km). These distances were lower than those from a previous study for hives located in a rural area 4.5 km away. Honey bees are very sensitive to foraging economics and foragers make waggle dances only after visiting high-quality feeding locations. Low distances advertised by dances, therefore, indicate sufficient forage nearby and show that urban areas can support honey bees year round. As a corollary, however, urban bees may provide little pollination service to agriculture especially in spring, which had the lowest foraging distances and is when the most economically important animal-pollinated UK crops, apple and oilseed rape, are in bloom.

Urban beekeeping is becoming more popular in the UK. One of the challenges faced by urban beekeepers is finding a suitable apiary location. Honey bees are often perceived as a nuisance, mainly due to their stinging behaviour. Here, we experimentally test the assumption that barriers around an apiary such as walls or fences, force the bees to fly above human height, thereby reducing collisions with people and, consequently, stinging. The experiment was conducted in two apiaries using two common types of barrier: a lattice fence (trellis) and hedge. Barriers were 2 m high, which is taller than > 99% of humans and is also the maximum height allowed by UK planning regulations for garden fences or walls. We found that barriers were effective at both raising the mean honey bee flight height and reducing stinging. However, the effects were only seen when the barrier had been in place for a few days, not immediately after the barrier was put in place. Although this raises interesting questions regarding honey bee navigation and memory, it is not a problem for beekeepers, as any barrier placed around an apiary will be permanent. The effect of the barriers on raising bee flight height to a mean of c. 2.2-2.5 m was somewhat weak and inconsistent, probably because the bees flew high, mean of c. 1.6-2.0 m, even in the absence of a barrier. As barriers can also reduce wind exposure, improve security and are inexpensive, we recommend their use around urban apiaries in places such as private gardens or allotments, where nuisance to humans is likely to be a problem.

1. Pollinating insects are globally declining, with one of the main causes being the loss of flowers. With the value of countryside reducing, urban areas, particularly gardens, are increasingly recognized as of benefit to wildlife, including flower-visiting insects.
2. Many gardeners specifically select plant varieties attractive to wildlife. Given the wide public interest, many lists of recommended varieties have been produced by both amateurs and professional organizations, but appear not to be well grounded in empirical data. These lists, however, are not without merit and are an obvious starting point. There is clearly a need to put the process onto a firmer footing based more on data and less on opinion and general experience.
3. We collected data over two summers by counting flower-visiting insects as they foraged on 32 popular summer-flowering garden plant varieties in a specially planted experimental garden, with two smaller additional gardens set up in year two to check the generality of the results. With many thousands of plant varieties available to gardeners in the United Kingdom, and other countries or regions, it would have been an impossible task to make a comprehensive survey resulting in a complete and authoritative list.
4. Our results are valuable and encouraging. Garden flowers attractive to the human eye vary enormously, approximately 100-fold, in their attractiveness to insects. Insects, especially bees and hover flies, can be attracted in large numbers with clear differences in the distribution of types attracted by different varieties.
5. Our results clearly show that there is a great scope for making gardens and parks more bee- and insect-friendly by plant selection. Horticulturally modified plant varieties created by plant breeding, including hybrids, are not necessarily less attractive to insects and in some cases are more attractive than their wild-type counterparts. Importantly, all the plants we compared were considered highly attractive to humans, given that they are widely sold as ornamental garden plants.
6. Helping insect pollinators in gardens does not involve extra cost or gardening effort, or loss of aesthetic attractiveness. Furthermore, the methods of quantifying insect-friendliness of plant varieties trialled in this study are relatively simple and can form the basis of further research, including ‘citizen science’.

1. Ivy (Hedera helix and H. hibernica) is a common autumn-flowering plant found in Europe, North Africa, Macaronesia and Asia. Here, we use five complementary approaches (pollen trapping, nectar refractometry, local and regional surveys of insects foraging on ivy flowers, local survey of ivy abundance) to evaluate its importance to the honey bee (Apis mellifera) and other flower-visiting insects in Sussex, England.
2. Pollen trapping at six hives in two locations showed that an average 89% of pollen pellets collected by honey bees in the autumn were from ivy.
3. Observations of foraging honey bees on ivy showed that ivy nectar is an even greater target than pollen, as 80% were collecting only nectar. Refractometry of samples from ivy flowers and from honey bees foraging on ivy showed that ivy nectar is rich in sugar, 49% w/w.
4. Surveys showed that the main insect taxa foraging on ivy were honey bees (21%), bumble bees (Bombus spp., 3%), ivy bees (Colletes hederae, 3%), common wasps (Vespula vulgaris, 13%), hover flies (Syrphidae, 27%), other flies (29%) and butterflies (4%). The surveys also showed significant temporal and spatial variation in taxon abundance and proportion.
5. A survey showed that ivy was very abundant on a small scale in both rural and urban areas, being present in 10/10 and 6/10 0.2 × 0.2 km samples within two 4 × 4 km areas respectively.
6. The results show that ivy should probably be considered a keystone species with a high value in the conservation of flower-visiting insects in autumn.

1. We examined the effects of reduced mowing on wildflower bloom and flower-visiting insects in the Saltdean Oval, a 6 ha suburban public park in Saltdean, UK.
2. In 2012, a novel management regime was initiated in which approximately half the grass area was left uncut, with the plan being to mow it once per year in autumn. In spring 2013, we set up four blocks, with each block subdivided into four 30 × 5 m strips treated under different mowing regimes: (a) regular mowing every 2 weeks all spring and summer, (b) regular mowing until 2 June, (c) regular mowing until 5 July, and (d) no mowing.
3. The abundance of both flowers and flower-visiting insects increased significantly with reduced mowing, being ca. 3 and 5 times greater in (d) than (a), respectively, with (b) and (c) intermediate. Mowing intensity, however, had a weak effect on wildflower species richness, which was only lower in (a).
4. A 1 km bee, butterfly, and moth transect walk, 500 m within the long grass part of the park and 500 m within the short, recorded ca. 50× greater insect abundance in the long grass.
5. A questionnaire of public opinion found that 97% of park visitors favoured encouraging insects and wildflowers. In terms of enjoyment of the park, 26% said that it had increased, 64% said that it stayed the same, and 10% said that it decreased.
6. These results present an encouraging example of a potential win–win situation in urban land management change, where the interests of humans and wildlife are aligned, thereby making the goals of conservation easier to achieve.

Pollinators are in global decline. One of the few ways in which the general public can help is by cultivating ornamental garden plants that attract pollinators by producing nectar, pollen, or both. Advice in the form of lists of recommended plants is available, but how good are these recommendations? Here, we overview a sample of 15 such lists and discuss their strengths and weaknesses. In particular, we found that the range of the number of plant genera per list was large (29–257) and that there was rather little overlap in the recommendations, even among lists addressing the same geographic region (e.g., Britain or North America). Furthermore, the lists often included poor recommendations, omitted many good plants, lacked detail, and were almost invariably based on their authors’ general expertize rather than on empirical data. Nevertheless, some advice given in the lists was good, because these recommendations were presumably backed by personal observations and less formally gathered data. The lists were also very appealing to the public, which makes them an excellent tool in communication and a useful starting point for further research.

Previous studies investigating the effect of flower patch size on insect flower visitation rate have compared relatively large patches (10–1000s m2) and have generally found a negative relationship per unit area or per flower. Here, we investigate the effects of patch size on insect visitation in patches of smaller area (range c. 0.1–3.1 m2), which are of particular relevance to ornamental flower beds in parks and gardens. We studied two common garden plant species in full bloom with 6 patch sizes each: borage (Borago officinalis) and lavender (Lavandula × intermedia ‘Grosso’). We quantified flower visitation by insects by making repeated counts of the insects foraging at each patch. On borage, all insects were honey bees (Apis mellifera, n = 5506 counts). On lavender, insects (n = 737 counts) were bumble bees (Bombus spp., 76.9%), flies (Diptera, 22.4%), and butterflies (Lepidoptera, 0.7%). On both plant species we found positive linear effects of patch size on insect numbers. However, there was no effect of patch size on the number of insects per unit area or per flower and, on lavender, for all insects combined or only bumble bees. The results show that it is possible to make unbiased comparisons of the attractiveness of plant species or varieties to flower-visiting insects using patches of different size within the small scale range studied and make possible projects aimed at comparing ornamental plant varieties using existing garden flower patches of variable area.

Social insects often respond to signals and cues from nest-mates, and these responses may include changes in the information they, in turn, transmit. During foraging, Lasius niger deposits a pheromone trail to recruit nestmates, and ants that experience trail crowding deposit pheromone less often. Less studied, however, is the time taken for signalling to revert to baseline levels after conditions have returned to baseline levels. In this paper we study the behaviour of L. niger foragers on a trail in which crowding is simulated by using dummy ants — black glass beads coated in nestmate cuticular hydrocarbons. Ants were allowed to make four repeat visits to a feeder with dummy ants, and thus crowding, being present on the trail on all visits (CCCC), none (UUUU) or only the first two (CCUU). If dummy ants were always present (CCCC), pheromone deposition probability was low in the first two visits (54% of ants deposited pheromone) and remained low in visits 3 and 4 (51%). If dummy ants were never present (UUUU) pheromone deposition probability was high in the first two visits (93%) and remained high in visits 3 and 4 (83%). If dummy ants were present on the first two visits but removed on the second two visits (CCUU) pheromone deposition probability was low in the first two visits (61%) but rose in the second two visits (69%). This demonstrates that even after pheromone deposition has been down-regulated due to crowding in the first two visits, it is rapidly up-regulated when crowding is reduced, although it does not immediately return to the base line level.

House flies (Musca domestica L.) are key pests of poultry and are managed worldwide with a variety of insecticides. However, extensive and injudicious use of insecticides has led to the development of resistance in many insect pests. Insecticide mixtures can increase the efficacy of the product and/or delay the development of resistance, thus making them a useful tool for pest control. In addition to their efficacy when used alone, mixtures of emamectin benzoate, spinosad or nitenpyram with either lambda-cyhalothrin or profenofos were assessed against a susceptible strain of house flies collected from poultry farms. The analysis revealed that combination indices for lambda-cyhalothrin/emamectin benzoate and lambda-cyhalothrin/spinosad mixtures were significantly >1, indicating an antagonistic effect. In contrast, the indices for all the tested ratios of lambda-cyhalothrin/nitenpyram were significantly < 1, suggesting a synergistic effect. For mixtures containing profenofos, combination indices varied significantly. For example, synergism was observed in a 1:1 mixture containing lambda-cyhalothrin and emamectin benzoate, but antagonism occurred when a higher proportion of the latter insecticide was used. Conversely, synergism in a mixture containing profenofos and nitenpyram was observed only when a lower proportion of the second insecticide was used. The present results suggest that ideal insecticide mixtures must be empirically determined and alternative strategies such as mosaics, rotations and cultural control should be considered for the management of the house fly.

Even as demand for their services increases, honey bees (Apis mellifera) and other pollinating insects continue to decline in Europe and North America. Honey bees face many challenges, including an issue generally affecting wildlife: landscape changes have reduced flower-rich areas. One way to help is therefore to supplement with flowers, but when would this be most beneficial? We use the waggle dance, a unique behaviour in which a successful forager communicates to nestmates the location of visited flowers, to make a 2-year survey of food availability. We “eavesdropped” on 5097 dances to track seasonal changes in foraging, as indicated by the distance to which the bees as economic foragers will recruit, over a representative rural-urban landscape. In year 3, we determined nectar sugar concentration. We found that mean foraging distance/area significantly increase from springs (493 m, 0.8 km2) to summers (2156 m, 15.2 km2), even though nectar is not better quality, before decreasing in autumns (1275 m, 5.1 km2). As bees will not forage at long distances unnecessarily, this suggests summer is the most challenging season, with bees utilizing an area 22 and 6 times greater than spring or autumn. Our study demonstrates that dancing bees as indicators can provide information relevant to helping them, and, in particular, can show the months when additional forage would be most valuable.

Since 1994, more than €41 billion has been spent in the European Union on agri-environment schemes (AESs), which aim to mitigate the effects of anthropomorphic landscape changes via financial incentives for land managers to encourage environmentally friendly practices [1, 2, 3, 4, 5, 6]. Surprisingly, given the substantial price tag and mandatory EU member participation [2], there is either a lack of [1] or mixed [1, 2, 7] evidence-based support for the schemes. One novel source of data to evaluate AESs may be provided by an organism that itself may benefit from them. Honeybees (Apis mellifera), important pollinators for crops and wildflowers [8, 9], are declining in parts of the world from many factors, including loss of available forage from agricultural intensification [10, 11, 12, 13]. We analyzed landscape-level honeybee foraging ecology patterns over two years by decoding 5,484 waggle dances from bees located in the center of a mixed, urban-rural 94 km2 area, including lands under government-funded AESs. The waggle dance, a unique behavior performed by successful foragers, communicates to nestmates the most profitable foraging locations [14, 15, 16]. After correcting for distance, dances demonstrate that honeybees possess a significant preference for rural land managed under UK Higher Level AESs and a significant preference against rural land under UK Organic Entry Level AESs. Additionally, the two most visited areas contained a National and Local Nature Reserve, respectively. Our study demonstrates that honeybees, with their great foraging range and sensitive response to forage quality, can be used as bioindicators to monitor large areas and provide information relevant to better environmental management.

Recent laboratory based studies have demonstrated adverse sub-lethal effects of neonicotinoid insecticides on honey bees and bumble bees, and these studies have been influential in leading to a European Union moratorium on the use of three neonicotinoids, clothianidin, imidacloprid, and thiamethoxam on “bee attractive” crops. Yet so far, these same effects have not been observed in field studies. Here we review the three key dosage factors (concentration, duration and choice) relevant to field conditions, and conclude that these have probably been over estimated in many laboratory based studies.

In the honey bee, hygienic behaviour, the removal of dead or diseased brood from capped cells by workers, is a heritable trait that confers colony-level resistance against brood diseases. This behaviour is quite rare. Only c. 10% of unselected colonies show high levels of hygiene. Previous studies suggested that hygiene might be rare because it also results in the removal of healthy brood, thereby imposing an ongoing cost even when brood diseases are absent. We tested this hypothesis by quantifying hygienic behaviour in 10 colonies using a standard technique, the freeze-killed brood (FKB) bioassay. At the same time, we also quantified the removal of untreated brood. The study colonies showed a wide range in hygienic behaviour, removing 19.7–100% of the FKB. The removal of untreated brood ranged from 2% to 44.4%. However, there was no correlation between the two removal rates for any of the four age groups of untreated brood studied (eggs, young larvae, older larvae from uncapped cells and larvae/pupae from capped cells). These results do not support the cost-to-healthy-brood hypothesis for the rarity of hygienic behaviour.

Hygienic behaviour in the honey bee, Apis mellifera, is the uncapping and removal of dead, diseased or infected brood from sealed cells by worker bees. We determined the effect of hygienic behaviour on varroa population growth and incidence of deformed wing virus (DWV), which can be transmitted by varroa. We treated 42 broodless honey bee colonies with oxalic acid in early January 2013 to reduce varroa populations to low levels, which we quantified by extracting mites from a sample of worker bees. We quantified varroa levels, again when the colonies were broodless, 48 weeks later. During the summer the hygienic behaviour in each colony was quantified four times using the Freeze Killed Brood (FKB) removal assay, and ranged from 27.5 % to 100 %. Varroa population increased greatly over the season, and there was a significant negative correlation between varroa increase and FKB removal. This was entirely due to fully hygienic colonies with >95 % FKB having only 43 % of the varroa build up of the less hygienic colonies.None of the 14 colonies with >80 % FKB removal had overt symptoms of DWV, whilst 36 % of the less hygienic colonies did. Higher levels of FKB removal also correlated significantly with lower numbers of DWV RNA copies in worker bees, but not in varroa mites. On average, fully hygienic colonies had c. 10,000 times less viral RNA than less hygienic colonies.

Neonicotinoid insecticides used to treat the seeds of bee-attractive crops occur in trace amounts in nectar and pollen. Possible harm to bees has resulted in the European Commission imposing a precautionary two-year moratorium on the use of neonicotinoids on bee-attractive crops from 2013. Recent laboratory and semi-field studies on colony-level effects of neonicotinoids assumed exclusive or near-exclusive levels of colony foraging on a treated crop. But is this a realistic assumption? Six honey bee (Apis mellifera) colonies were monitored over two springs (April–May 2011/12) in two neighbouring locations (urban and rural) in and near Brighton, UK, to quantify foraging on oilseed rape, the most widespread bee-attractive crop in the UK, by decoding waggle dances and trapping pollen. The study area was representative of the UK agricultural landscape in that the percentage area cover of the blooming oilseed rape fields around the rural location was similar to the national average (3.3–3.9% vs 3.1%). The amount of foraging on oilseed rape fields, as indicated by dance decoding, was variable, but low, 0–0.02% for the urban and 2–26% for the rural location. Almost all foraging, 91–99%, was within 2 km, even though honey bees can forage at distances of over 10 km. Pollen trapping in 2012 supported the dance decoding results, with oilseed rape pollen comprising 14% of pollen pellets collected by foragers from rural and 4% from urban hives. The results of this study have implications for policy as they cast doubt on the generality of some previous studies on colony-level effects on social bees conducted in laboratory and semi-field settings.

Trail pheromones do more than simply guide social insect workers from point A to point B. Recent research has revealed additional ways in which they help to regulate colony foraging, often via positive and negative feedback processes that influence the exploitation of the different resources that a colony has knowledge of. Trail pheromones are often complementary or synergistic with other information sources, such as individual memory. Pheromone trails can be composed of two or more pheromones with different functions, and information may be embedded in the trail network geometry. These findings indicate remarkable sophistication in how trail pheromones are used to regulate colony-level behavior, and how trail pheromones are used and deployed at the individual level.

To investigate the distances at which honey bee foragers collect nectar and pollen, we analysed 5,484 decoded waggle dances made to natural forage sites to determine monthly foraging distance for each forage type. Firstly, we found significantly fewer overall dances made for pollen (16.8 %) than for non-pollen, presumably nectar (83.2 %; P < 2.2 × 10−23). When we analysed distance against month and forage type, there was a significant interaction between the two factors, which demonstrates that in some months, one forage type is collected at farther distances, but this would reverse in other months. Overall, these data suggest that distance, as a proxy for forage availability, is not significantly and consistently driven by need for one type of forage over the other.

Context plays an important role in a discriminator's ability to make appropriate recognition decisions, such as accepting what is acceptable and rejecting what is not acceptable. Previously it was shown that in both honey bees and stingless bees, discriminating workers (guards) make more errors towards conspecific non-nestmates when the guards are removed from the natural hive entrance. However, it may be that guards, in addition to making incorrect recognition decisions, also may adopt non-guarding behaviours. Here, we tested honey bee guards in two contexts (natural versus unnatural) against five types of introduced arthropods (conspecific nestmates and non-nestmates; allospecific wasps, beetles and woodlice), which should be rejected without error. We scored a guard's response as accept, reject, avoid and ignore. Total errors significantly increased from natural to unnatural contexts. Specifically, guards were significantly more likely to make an acceptance error, guarding and accepting both conspecific and allospecific non-nestmates, in the unnatural context. Importantly, guards were significantly more likely to adopt a non-guarding behaviour in the unnatural context, which usually involved ignoring or avoiding, where a guard makes contact but then immediately retreats, the introduced arthropod. Overall, these data demonstrate the context is important. Removing a guard from the home that it protects elicits either incorrect discrimination or, additionally, a complete lack of discriminator behaviour altogether.

Competition between managed honeybees and wild pollinators is thought to be a key factor in structuring foraging communities on flowers. The majority of studies have focused on impacts such as floral visitation rates and resource overlap. However, direct measurement of fitness is required to fully assess the impacts of competition. We compared in 2 years the weight and reproductive success of bumblebee colonies located at two sites that were either close to or far from a large honeybee apiary, and which were located in the same landscape and with access to similar floral resources. We found that bumblebee colonies located at the site near the honeybee apiary gained less weight, and produced fewer and smaller queens, in both years than colonies at the site far from the apiary. The ratio of queen weight/size was lower in the colonies near honeybees in 1 year, while males were smaller and offspring sex ratio more male biased in colonies close to honeybees than in those far from honeybees. Proximity to managed honeybee hives was therefore associated with significantly reduced fitness of bumblebee colonies, but studies from many more sites are needed to confirm the effect.

Detecting and responding to threats are of prime importance for social species which need to be able to distinguish nestmates from intruders to protect the resources of their colony. However, individuals may differ in their propensity to recognise threats due to factors, often intercorrelated, such as caste, age and experience, and the ability to separate these is important for understanding why behaviours are expressed. Here, we use leaf-cutting ants in a controlled behavioural assay to tease apart the factors which likely affect threat response behaviours in social insect workers. We show that foraging workers respond to threats more readily than do within-nest workers. The response of all workers was greater towards more foreign stimuli—nestmates rarely stimulated a response, whereas ants of a different genus stimulated a response in most cases. We show that age and experience act separately to increase an individual’s ability to perceive the threat. This suggests that where multiple, compounding factors affect the expression of certain behaviours it is important to realise that these factors can also have independent effects, particularly those which correlate with age. Separating the influence of correlating factors experimentally, as shown here, is particularly useful for understanding why individuals may differ in their behavioural profile.

Immunosenescence, the systemic reduction of immune efficiency with age, is increasingly recognised as having important implications for host-parasite dynamics. Changes in the immune response can impact on the ability of an individual to resist or moderate parasite infection, depending on how and when it encounters a parasite challenge. Using the European honey bee Apis mellifera and its microsporidian parasite Nosema ceranae, we investigated the effects of host age on the ability to resist parasite infection and on baseline immunocompetence, assessed by quantifying constitutive (PO) and potential levels (PPO) of the phenoloxidase immune enzyme as general measures of immune function. There was a significant correlation between the level of general immune function and infection intensity, but not with survival, and changes in immune function with age correlated with the ability of individuals to resist parasite infection. Older individuals had better survival when challenged with a parasite than younger individuals, however they also had more intense infections and lower baseline immunocompetence. The ability of older individuals to have high infection intensities yet live longer, has potential consequences for parasite transmission. The results highlight the need to consider age in host-parasite studies and show the importance of choosing the correct measure when assaying invertebrate immunity.

Transmission is a key determinant of parasite fitness, and understanding the dynamics of transmission is fundamental to the ecology and evolution of host–parasite interactions. Successful transmission is often reliant on contact between infected individuals and susceptible hosts. The social insects consist of aggregated groups of genetically similar hosts, making them particularly vulnerable to parasite transmission. Here we investigate how the ratio of infected to susceptible individuals impacts parasite transmission, using the honey bee, Apis mellifera and its microsporidian parasite Nosema ceranae. We used 2 types of infected hosts found simultaneously in colonies; sterile female workers and sexual males. We found a higher ratio of infected to susceptible individuals in groups resulted in a greater proportion of susceptibles becoming infected, but this effect was non-linear and interestingly, the ratio also affected the spore production of infected individuals. The transmission level was much greater in an experiment where the infected individuals were drones than in an experiment where they were workers, suggesting drones may act as intracolonial ‘superspreaders’. Understanding the subtleties of transmission and how it is influenced by the phenotype of the infected/susceptible individuals is important for understanding pathogen transmission at population level, and for optimum targeting of parasite control strategies.

The ability of an organism to detect threats is fundamental to mounting a successful defense and this is particularly important when resisting parasites. Early detection of parasites allows for initiation of defense mechanisms, which are vital in mitigating the cost of infection and are likely to be especially important in social species, particularly those whose life history makes parasite pressure more significant. However, understanding the relative strength of behavioral responses in different species and situations is still limited. Here, we test the response of individual ants to fungal parasites in 3 different contexts, for 4 ant species with differing life histories. We found that ants from all 4 species were able to detect fungi on their food, environment, and nest mates and initiate avoidance or upregulate grooming behaviors accordingly to minimize the threat to themselves and the colony. Individuals avoided fungal-contaminated surfaces and increased grooming levels in response to fungal-contaminated nest mates. Ants from all species responded qualitatively in a similar way although the species differed quantitatively in some respects that may relate to life-history differences. The results show that ants of multiple species are capable of recognizing fungal threats in various contexts. The recognition of parasite threats may play an important role in enabling ant colonies to deal with the ever-present threat from disease.

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders.

Drug discovery is a constantly developing and expanding area of research. Developed to provide a comprehensive guide, the Handbook of Medicinal Chemistry covers the past, present and future of the entire drug development process. Highlighting the recent successes and failures in drug discovery, the book will help readers to understand the factors governing modern drug discovery from the initial concept through to a marketed medicine. With chapters covering a wide range of topics from drug discovery processes and optimization, development of synthetic routes, pharmaceutical properties and computational biology, the handbook aims to help medicinal chemists to apply their academic understanding to every aspect of drug discovery. Each chapter includes expert advice to not only provide a rigorous understanding of the principles being discussed, but to provide useful hints and tips gained from within the pharmaceutical industry. This expertise, combined with project case studies, highlighting and discussing all areas of successful projects, make this an essential handbook for all those involved in pharmaceutical development. A free app has been created in collaboration with the editors of the book. The Medicinal Chemistry Toolkit provides a suite of resources to support the day to day work of a medicinal chemist.

Low-level social aggression is a conspicuous feature of cooperative animal societies, but its precise function is usually unclear. One long-standing hypothesis is that aggressive displays by dominant individuals serve to reduce uncertainty about relative strength and deter subordinates from starting fights that they are unlikely to win. However, most formal theoretical models of this idea do not consider how the credibility of deterrent signals might change over time in social groups. We developed a simple model of dominant aggression as a deterrent signal, which takes into account how credibility changes over time and how selection should act on receiver memory. We then carried out an experimental test of the predictions of our model on a field population of the paper wasp, Polistes dominulus. The match between our theoretical and empirical results suggests that low-level social aggression can help to maintain the stability and productivity of cooperative associations in this species. Moreover, our work suggests that rates of aggression in animal societies and the robustness of social memories are likely to be intimately related.

Using simple stochastic models, we discuss how cooperative breeders, especially wasps and bees, can improve their productivity by reducing foraging work. In a harsh environment, where foraging is the main cause of mortality, such breeders achieve greater productivity by reducing their foraging effort below full capacity, and they may thrive by adopting cooperative breeding. This could prevent the population extinction of cooperative breeders under conditions where a population of lone breeders cannot be maintained.

Relatedness is predicted to be a key determinant of cooperative behavior, but kin discrimination within social insect colonies is surprisingly rare. A lack of reliable cuticular hydrocarbon (CHC) cues is thought to be responsible, but here we show that in a high-profile paper wasp model, kin recognition cues are available for some individuals that found nests with nonrelatives. Thus, unrelated Polistes dominulus helpers could potentially recognize themselves as such. On this basis, we reanalyzed a behavioral data set to investigate whether foraging effort, defense contributions and aggression toward nest mates might thus reflect CHC profiles. Both foraging behavior and aggression varied with genetic relatedness, but genetic relatedness itself was a better predictor of this variation than differences in CHC profiles. We propose that wasps use specific components of the CHC profile, the identity of which is as yet unknown, to identify relatives among nest mates. Our data provide the first evidence of within-nest kin discrimination in primitively eusocial wasps but leave open the question of which cues are responsible.

Queues formed by social wasps to inherit the dominant position in the nest are analyzed by using a transient quasi-birth-and-death (QBD) process. We show that the extended nest lifespan due to division of labor between queen and helpers has a big impact on nest productivity.

Recent theory and empirical studies of avian biparental systems suggest that animals resolve conflict over parental care via a process of behavioral negotiation or “rules for responding.” Less is known, however, about whether negotiation over helping effort occurs in cooperatively breeding animal societies or whether behavioral negotiation requires a relatively large brain. In this study, we tested whether negotiation over help occurs in a social insect, the paper wasp Polistes dominulus, by recording individual responses to both observed and experimentally induced foraging returns by other group members. In our experiments, we manipulated food delivery to the nest in 2 ways: 1) by catching departing foragers and giving them larval food to take back to the nest and 2) by giving larval food directly to wasps on the nest, which they then fed to larvae, so increasing food delivery independently of helper effort. We found no evidence from Experiment 1 that helpers adjusted their own foraging effort according to the foraging effort of other group members. However, when food was provided directly to the nest, wasps did respond by reducing their own foraging effort. One interpretation of this result is that paper wasp helpers adjust their helping effort according to the level of offspring need rather than the work rate of other helpers. Negotiation based on indicators of demand rather than work rate is a likely mechanism to resolve conflict over investment in teams where helpers cannot observe each other’s work rate directly, as is commonly the case in insect and vertebrate societies.

Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene.

IMPORTANCE The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the physiological regulation of cell genes by EBNA-2.

Background Most of the water on Earth is seawater, each kilogram of which contains about 35 g of salts, and yet most plants cannot grow in this solution; less than 0·2 % of species can develop and reproduce with repeated exposure to seawater. These ‘extremophiles’ are called halophytes.

Scope Improved knowledge of halophytes is of importance to understanding our natural world and to enable the use of some of these fascinating plants in land re-vegetation, as forages for livestock, and to develop salt-tolerant crops. In this Preface to a Special Issue on halophytes and saline adaptations, the evolution of salt tolerance in halophytes, their life-history traits and progress in understanding the molecular, biochemical and physiological mechanisms contributing to salt tolerance are summarized. In particular, cellular processes that underpin the ability of halophytes to tolerate high tissue concentrations of Na+ and Cl−, including regulation of membrane transport, their ability to synthesize compatible solutes and to deal with reactive oxygen species, are highlighted. Interacting stress factors in addition to salinity, such as heavy metals and flooding, are also topics gaining increased attention in the search to understand the biology of halophytes.

Conclusions Halophytes will play increasingly important roles as models for understanding plant salt tolerance, as genetic resources contributing towards the goal of improvement of salt tolerance in some crops, for re-vegetation of saline lands, and as ‘niche crops’ in their own right for landscapes with saline soils.

Background and Aims Osmolytes are low-molecular-weight organic solutes, a broad group that encompasses a variety of compounds such as amino acids, tertiary sulphonium and quaternary ammonium compounds, sugars and polyhydric alcohols. Osmolytes are accumulated in the cytoplasm of halophytic species in order to balance the osmotic potential of the Na+ and Cl− accumulated in the vacuole. The advantages of the accumulation of osmolytes are that they keep the main physiological functions of the cell active, the induction of their biosynthesis is controlled by environmental cues, and they can be synthesized at all developmental stages. In addition to their role in osmoregulation, osmolytes have crucial functions in protecting subcellular structures and in scavenging reactive oxygen species.

Scope This review discusses the diversity of osmolytes among halophytes and their distribution within taxonomic groups, the intrinsic and extrinsic factors that influence their accumulation, and their role in osmoregulation and osmoprotection. Increasing the osmolyte content in plants is an interesting strategy to improve the growth and yield of crops upon exposure to salinity. Examples of transgenic plants as well as exogenous applications of some osmolytes are also discussed. Finally, the potential use of osmolytes in protein stabilization and solvation in biotechnology, including the pharmaceutical industry and medicine, are considered.

Background Halophytes are the flora of saline soils. They adjust osmotically to soil salinity by accumulating ions and sequestering the vast majority of these (generally Na+ and Cl−) in vacuoles, while in the cytoplasm organic solutes are accumulated to prevent adverse effects on metabolism. At high salinities, however, growth is inhibited. Possible causes are: toxicity to metabolism of Na+ and/or Cl− in the cytoplasm; insufficient osmotic adjustment resulting in reduced net photosynthesis because of stomatal closure; reduced turgor for expansion growth; adverse cellular water relations if ions build up in the apoplast (cell walls) of leaves; diversion of energy needed to maintain solute homeostasis; sub-optimal levels of K+ (or other mineral nutrients) required for maintaining enzyme activities; possible damage from reactive oxygen species; or changes in hormonal concentrations.

Scope This review discusses the evidence for Na+ and Cl− toxicity and the concept of tissue tolerance in relation to halophytes.

Conclusions The data reviewed here suggest that halophytes tolerate cytoplasmic Na+ and Cl− concentrations of 100–200 mm, but whether these ions ever reach toxic concentrations that inhibit metabolism in the cytoplasm or cause death is unknown. Measurements of ion concentrations in the cytosol of various cell types for contrasting species and growth conditions are needed. Future work should also focus on the properties of the tonoplast that enable ion accumulation and prevent ion leakage, such as the special properties of ion transporters and of the lipids that determine membrane permeability.

The world's food production will need to increase by up to 70% by 2050 to match the predicted population growth. Achieving this goal will be challenging due to the decreased availability of arable land, resulting from urbanization and land degradation. Soil salinity is a major factor contributing to the latter process. While some improvement in crop yields in saline soils may be achieved as a consequence of single gene transfers, the real progress may be achieved only via a painfully slow “pyramiding” of essential physiological traits. Given the time constraints, a safer solution to meet the 2050 challenge may be to find alternative crop and forage species for farming in salt-affected conditions and to restore salt-affected areas. This review focuses on the suitability of halophytes to become important components of 21st century farming systems. We provide a comprehensive summary of the current use of halophytes for human food consumption, for forage and animal feeds, as oilseed and energy crops, and for desalination and phytoremediation purposes. We argue that the use of halophytes may be a viable commercial alternative to ease pressure on the requirement of good quality land and water for conventional cropping systems and the utilization of land degraded by salinity.

With the advent of Next Generation Sequencing the identification of mutations in the genomes of healthy and diseased tissues has become commonplace. While much progress has been made to elucidate the aetiology of disease processes in cancer, the contributions to disease that many individual mutations make remain to be characterised and their downstream consequences on cancer phenotypes remain to be understood. Missense mutations commonly occur in cancers and their consequences remain challenging to predict. However, this knowledge is becoming more vital, for both assessing disease progression and for stratifying drug treatment regimes. Coupled with structural data, comprehensive genomic databases of mutations such as the 1000 Genomes project and COSMIC give an opportunity to investigate general principles of how cancer mutations disrupt proteins and their interactions at the molecular and network level. We describe a comprehensive comparison of cancer and neutral missense mutations; by combining features derived from structural and interface properties we have developed a carcinogenicity predictor, InCa (Index of Carcinogenicity). Upon comparison with other methods, we observe that InCa can predict mutations that might not be detected by other methods. We also discuss general limitations shared by all predictors that attempt to predict driver mutations and discuss how this could impact high-throughput predictions. A web interface to a server implementation is publicly available at http://inca.icr.ac.uk/.

The DNA damage response (DDR) is essential for maintaining the genomic integrity of the cell, and its disruption is one of the hallmarks of cancer. Classically, defects in the DDR have been exploited therapeutically in the treatment of cancer with radiation therapies or genotoxic chemotherapies. More recently, protein components of the DDR systems have been identified as promising avenues for targeted cancer therapeutics. Here, we present an in-depth analysis of the function, role in cancer and therapeutic potential of 450 expert-curated human DDR genes. We discuss the DDR drugs that have been approved by the US Food and Drug Administration (FDA) or that are under clinical investigation. We examine large-scale genomic and expression data for 15 cancers to identify deregulated components of the DDR, and we apply systematic computational analysis to identify DDR proteins that are amenable to modulation by small molecules, highlighting potential novel therapeutic targets.

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete, therefore the aim of this research was to investigate on recently identify factors that affect the susceptibility of insects to Bt (protein levels, midgut bacteria and mutations on the ABCC2 transporter) in five brazilian populations of Plutella xylostella larvae and to overcome the mechanism of resistance. The analyses were made in about 60 guts extracts in 1 ml of 50% glycerol solution using the following process: gut analysis (total protein, total proteas, toxin digestion and esterase activities); gut bacteria analysis; and ABCC2 gene analysis. There were not differences in the amount of esterases between the populations, therefore we cannot associate esterase level with resistance. Differences in composition of the larval gut microbiota were not correlated with resistance. It is unclear whether similar interactions occur in the field where the intestinal micrbiotas of lepidopteran larvae may be more complex than the apparent mono-associations described here. However it is possible that gut bacteria may present a novel target for the management of P. xylostella. The nature of the protective effect of gut bacteria is also unclear. Previous studies suggested that lactic acid bacteria may acidify the gut thereby decreasing host susceptibility to alkali-soluble Cry toxins such as CrylAc. There are no mutation in the ABCC2, accordingly there is no association of ABCC2 with Bt resistance.

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+.

Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3⇓–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca2+ entry through the channel itself (6⇓⇓⇓⇓⇓⇓–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14⇓–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca2+, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca2+ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca2+-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca2+ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20⇓⇓–23) compared with the piezo-driven glass rod (19, 24).

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca2+ chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current–voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.

The side effects of the current global use of pesticides on wildlife, particularly at higher levels of biological organization: populations, communities and ecosystems, are poorly understood (Köhler and Triebskorn 2013). Here, we focus on one of the problematic groups of agrochemicals, the systemic insecticides fipronil and those of the neonicotinoid family. The increasing global reliance on the partly prophylactic use of these persistent and potent neurotoxic systemic insecticides has raised concerns about their impacts on biodiversity, ecosystem functioning and ecosystem services provided by a wide range of affected species and environments. The present scale of use, combined with the properties of these compounds, has resulted in widespread contamination of agricultural soils, freshwater resources, wetlands, non-target vegetation and estuarine and coastal marine systems, which means that many organisms inhabiting these habitats are being repeatedly and chronically expose...

Since their discovery in the late 1980s, neonicotinoid pesticides have become the most widely used class of insecticides worldwide, with large-scale applications ranging from plant protection (crops, vegetables, fruits), veterinary products, and biocides to invertebrate pest control in fish farming. In this review, we address the phenyl-pyrazole fipronil together with neonicotinoids because of similarities in their toxicity, physicochemical profiles, and presence in the environment. Neonicotinoids and fipronil currently account for approximately one third of the world insecticide market; the annual world production of the archetype neonicotinoid, imidacloprid, was estimated to be ca. 20,000 tonnes active substance in 2010. There were several reasons for the initial success of neonicotinoids and fipronil: (1) there was no known pesticide resistance in target pests, mainly because of their recent development, (2) their physicochemical properties included many advantages over previous generations of insecticides (i.e., organophosphates, carbamates, pyrethroids, etc.), and (3) they shared an assumed reduced operator and consumer risk. Due to their systemic nature, they are taken up by the roots or leaves and translocated to all parts of the plant, which, in turn, makes them effectively toxic to herbivorous insects. The toxicity persists for a variable period of time—depending on the plant, its growth stage, and the amount of pesticide applied. A wide variety of applications are available, including the most common prophylactic non-Good Agricultural Practices (GAP) application by seed coating. As a result of their extensive use and physicochemical properties, these substances can be found in all environmental compartments including soil, water, and air. Neonicotinoids and fipronil operate by disrupting neural transmission in the central nervous system of invertebrates. Neonicotinoids mimic the action of neurotransmitters, while fipronil inhibits neuronal receptors. In doing so, they continuously stimulate neurons leading ultimately to death of target invertebrates. Like virtually all insecticides, they can also have lethal and sublethal impacts on non-target organisms, including insect predators and vertebrates. Furthermore, a range of synergistic effects with other stressors have been documented. Here, we review extensively their metabolic pathways, showing how they form both compound-specific and common metabolites which can themselves be toxic. These may result in prolonged toxicity. Considering their wide commercial expansion, mode of action, the systemic properties in plants, persistence and environmental fate, coupled with limited information about the toxicity profiles of these compounds and their metabolites, neonicotinoids and fipronil may entail significant risks to the environment. A global evaluation of the potential collateral effects of their use is therefore timely. The present paper and subsequent chapters in this review of the global literature explore these risks and show a growing body of evidence that persistent, low concentrations of these insecticides pose serious risks of undesirable environmental impacts.

Bumblebees are ecologically and economically important, and some species have suffered dramatic population declines. The absence of morphological diagnostic characters for the identification of some species creates difficulties for basic ecological studies, and for conservation management. The widespread and commercially exploited bumblebee subgenus Bombus sensu stricto contains a cryptic species complex, known as the lucorum complex, which in Europe comprises B. lucorum, B. cryptarum and B. magnus. Little is known about these species and much of what has been reported is likely to have suffered from incorrect identification. Although the lucorum complex as a whole is common in Great Britain, we aimed to determine whether the populations of the individual species are vulnerable and require conservation action. Using genetic methods to distinguish them, we determined the geographic distribution and abundance of the lucorum complex species in Great Britain, and assessed the extent of niche differentiation between these species. We detected major differences in the geographic range, forage use and sensitivity to summer temperatures of the three species. Bombus lucorum was found to have the broadest distribution and diet, being present throughout mainland Great Britain, whereas B. cryptarum and B. magnus were absent from large areas of central and southern England. Bombus cryptarum and B. magnus were more likely to be found at sites with lower summer temperatures. Bombus magnus, the least abundant species, was found to exhibit an unusually tight biotope association with heathland habitat. This has conservation implications for B. magnus given the current threats to this habitat type.

Agri-environment schemes have been implemented across the European Union in order to reverse declines in farmland biodiversity. To assess the impact of these schemes for bumblebees, accurate measures of their populations are required. Here, we compared bumblebee population estimates on 16 farms using three commonly used techniques: standardised line transects, coloured pan traps and molecular estimates of nest abundance. There was no significant correlation between the estimates obtained by the three techniques, suggesting that each technique captured a different aspect of local bumblebee population size and distribution in the landscape. Bumblebee abundance as observed on the transects was positively influenced by the number of flowers present on the transect. The number of bumblebees caught in pan traps was positively influenced by the density of flowers surrounding the trapping location and negatively influenced by wider landscape heterogeneity. Molecular estimates of the number of nests of Bombus terrestris and B. hortorum were positively associated with the proportion of the landscape covered in oilseed rape and field beans. Both direct survey techniques are strongly affected by floral abundance immediately around the survey site, potentially leading to misleading results if attempting to infer overall abundance in an area or on a farm. In contrast, whilst the molecular method suffers from an inability to detect sister pairs at low sample sizes, it appears to be unaffected by the abundance of forage and thus is the preferred survey technique.

We assessed the state of knowledge regarding the effects of large-scale pollution with neonicotinoid insecticides and fipronil on non-target invertebrate species of terrestrial, freshwater and marine environments. A large section of the assessment is dedicated to the state of knowledge on sublethal effects on honeybees (Apis mellifera) because this important pollinator is the most studied non-target invertebrate species. Lepidoptera (butterflies and moths), Lumbricidae (earthworms), Apoidae sensu lato (bumblebees, solitary bees) and the section “other invertebrates” review available studies on the other terrestrial species. The sections on freshwater and marine species are rather short as little is known so far about the impact of neonicotinoid insecticides and fipronil on the diverse invertebrate fauna of these widely exposed habitats. For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large-scale and wide ranging negative biological and ecological impacts on a wide range of non-target invertebrates in terrestrial, aquatic, marine and benthic habitats.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95,000 or 97,500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120,000) enzymes.

The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97,513 and an N-terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the ribA gene of Bacillus subtilis. The 1004-bp cysB--acn intergenic region contains several potential promoter and regulatory sequences. The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27-29% identity) and the isopropylmalate isomerases (20-21% identity) but it is most similar to the human iron-responsive-element-binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe-4S] centre are conserved in all of these proteins. Of the remaining 17 active-site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron-responsive-element-binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron-responsive-element-binding protein form a family of structurally related proteins, which does not include the Fe-S-containing fumarases. These relationships raise the possibility that the iron-responsive-element-binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron-responsive regulatory function.

Embryonic stem cells (ESCs) are maintained in an undifferentiated state through expression of the core transcriptional factors Nanog, Oct4, and Sox2. However, the epigenetic regulation of pluripotency is poorly understood. Differentiation of ESCs is accompanied by a global reduction of panacetylation of histones H3 and H4 suggesting that histone acetylation plays an important role in maintenance of ESC pluripotency. Acetylated lysine residues on histones are read by members of the bromodomain family that includes BET (bromodomain and extraterminal domain) proteins for which highly potent and selective inhibitors have been developed. In this study we demonstrate that the pan-BET bromodomain inhibitor JQ1 induces rapid spontaneous differentiation of murine ESCs by inducing marked transcriptional downregulation of Nanog as well as the stemness markers Lefty1 and Lefty2, but not Myc, often used as a marker of BET inhibitor activity in cancer. We show that the effects of JQ1 are recapitulated by knockdown of the BET family member BRD4 implicating this protein in Nanog regulation. These data are also supported by chromatin immunoprecipitation experiments which confirm BRD4 binding at the Nanog promoter that is known to require acetylation by the histone acetyltransferase MOF for transcriptional activity. In further support of our findings, we show that JQ1 antagonizes the stem cell-promoting effects of the histone deacetylase inhibitors sodium butyrate and valproic acid. Our data suggest that BRD4 is critical for the maintenance of ESC pluripotency and that this occurs primarily through the maintenance of Nanog expression.

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

Governments have committed to conserving ≥17% of terrestrial and ≥10% of marine environments globally, especially “areas of particular importance for biodiversity” through “ecologically representative” Protected Area (PA) systems or other “area-based conservation measures,” while individual countries have committed to conserve 3–50% of their land area. We estimate that PAs currently cover 14.6% of terrestrial and 2.8% of marine extent, but 59–68% of ecoregions, 77–78% of important sites for biodiversity, and 57% of 25,380 species have inadequate coverage. The existing 19.7 million km2 terrestrial PA network needs only 3.3 million km2 to be added to achieve 17% terrestrial coverage. However, it would require nearly doubling to achieve, cost-efficiently, coverage targets for all countries, ecoregions, important sites, and species. Poorer countries have the largest relative shortfalls. Such extensive and rapid expansion of formal PAs is unlikely to be achievable. Greater focus is therefore needed on alternative approaches, including community- and privately managed sites and other effective area-based conservation measures.

The function of pollen as a reward for foraging bees is little understood, though there is evidence to suggest that it can reinforce associations with visual and olfactory floral cues. Foraging bees do not feed on pollen, thus one could argue that it cannot serve as an appetitive reinforcer in the same way as sucrose. However, ingestion is not a critical parameter for sucrose reinforcement, since olfactory proboscis extension (PER) learning can be conditioned through antennal stimulation only. During pollen collection, the antennae and mouthparts come into contact with pollen, thus it is possible that pollen reinforces associative learning through similar gustatory pathways as sucrose. Here pollen was presented as the unconditioned stimulus (US), either in its natural state or in a 30% pollen-water solution, and was found to elicit proboscis extension following antennal stimulation. Control groups were exposed to either sucrose or a clean sponge as the US, or an unpaired presentation of the conditioned stimulus (CS) and pollen US. Despite steady levels of responding to the US, bees did not learn to associate a neutral odour with the delivery of a pollen reward, thus whilst pollen has a proboscis extension releasing function, it does not reinforce olfactory PER learning.

Stem cell research has been acclaimed to revolutionize the future of medicine, and to offer new treatments for previously incurable diseases. Despite years of research, however, the therapeutic potential of stem cell research has not yet been fully realized. By June 2014, the US Food and Drug Administration had approved only five stem cell-based medicinal products, all of which cord blood derived hematopoietic stem cell products for the cure of blood and immunological diseases. Anticipated treatments for cancer, neurodegenerative disorders, gastroenterological, myocardial, and other diseases are still far from routine applications. What are the reasons for the slow progress in the stem cell field, and the mismatch between public expectations and actual achievements?

This paper analyses the current problems of public and private banks, and gives two hybrid models which facilitate the cell exchange and allocate resource more efficiently. One is the 'public and private combination' model, and the other is the 'donatable family banking' model. It analyzes these models in terms of cell exchange, quality and financial consideration. This paper provides an alternative perspective on how the cord blood industry can cooperate with stem cell science.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

Stonebrood is a disease of honey bee larvae caused by fungi from the genus Aspergillus. As very few studies have focused on the epidemiological aspects of stonebrood and diseased brood may be rapidly discarded by worker bees, it is possible that a high number of cases go undetected. Aspergillus spp. fungi are ubiquitous and associated with disease in many insects, plants, animals and man. They are regarded as opportunistic pathogens that require immunocompromised hosts to establish infection. Microbiological studies have shown high prevalences of Aspergillus spp. in apiaries which occur saprophytically on hive substrates. However, the specific conditions required for pathogenicity to develop remain unknown. In this study, an apiary was screened to determine the prevalence and diversity of Aspergillus spp. fungi. A series of dose–response tests were then conducted using laboratory reared larvae to determine the pathogenicity and virulence of frequently occurring isolates. The susceptibility of adult worker bees to Aspergillus flavus was also tested. Three isolates (A. flavus, Aspergillus nomius and Aspergillus phoenicis) of the ten species identified were pathogenic to honey bee larvae. Moreover, adult honey bees were also confirmed to be highly susceptible to A. flavus infection when they ingested conidia. Neither of the two Aspergillus fumigatus strains used in dose–response tests induced mortality in larvae and were the least pathogenic of the isolates tested. These results confirm the ubiquity of Aspergillus spp. in the apiary environment and highlight their potential to infect both larvae and adult bees.

Remarkable variation exists in the distribution of reproduction (skew) among members of cooperatively breeding groups, both within and between species. Reproductive skew theory has provided an important framework for understanding this variation. In the primitively eusocial Hymenoptera, two models have been routinely tested: concessions models, which assume complete control of reproduction by a dominant individual, and tug-of-war models, which assume on-going competition among group members over reproduction. Current data provide little support for either model, but uncertainty about the ability of individuals to detect genetic relatedness and difficulties in identifying traits conferring competitive ability mean that the relative importance of concessions versus tug-of-war remains unresolved. Here, we suggest that the use of social parasitism to generate meaningful variation in key social variables represents a valuable opportunity to explore the mechanisms underpinning reproductive skew within the social Hymenoptera. We present a direct test of concessions and tug-of-war models in the paper wasp Polistes dominulus by exploiting pronounced changes in relatedness and power structures that occur following replacement of the dominant by a congeneric social parasite. Comparisons of skew in parasitized and unparasitized colonies are consistent with a tug-of-war over reproduction within P. dominulus groups, but provide no evidence for reproductive concessions.

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.

An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.

Budding yeast Mec1, a homolog of mammalian ATR/ATM, is an essential chromosome-based signal transduction protein. Mec1 is a key checkpoint regulator and plays a critical role in the maintenance of genome stability. Mec1 is also required for meiosis; loss of Mec1 functions leads to a number of meiotic defects including reduction in recombination, loss of inter-homolog bias, loss of crossover control, and failure in meiotic progression. Here we review currently available data on meiotic defects associated with loss of Mec1 functions and discuss the possibility that Mec1 may participate as a fundamentally positive player in coordinating and promoting basic meiotic chromosomal processes during normal meiosis.

Proliferating cells of Allium cepa L. roots became adapted to hypoxia (5% oxygen) and cold (10°C) by acquiring new steady-state kinetics of growth. The cell cycle time increased from the 17.6 h in control meristems up to 29.7 and 69.0 h under hypoxia and cold conditions, respectively. Acclimation of the proliferating cells was stress specific. No acclimation took place after 24 h of heat treatment (40°C). Under cold treatment, all cycle phases enlarged uniformly. However, under hypoxia, while the G1 and S cycle phases roughly doubled in their timing, the expected checkpoint-dependent lengthening of G2 did not take place. This failure in lengthening G2 in response to hypoxia correlated with a failure in the overinduction of a single peptide with a molecular mass of about 134 kDa which is among those recognised by an HSP90 antibody. Moreover, the presence of this large peptide of the HSP90 family proved to be a marker for cell proliferation. It was always absent from the contiguous differentiated cells of the root. Lastly, the mitochondrial chaperonin recognized by an HSP60 antibody in these roots not involved in photosynthesis was always higher in the proliferating than in the nonproliferating cells.

Hox genes encode a family of evolutionarily conserved transcription factors involved in
the activation of diverse cell differentiation programs along the antero-posterior axis of
animals. Hox gene expression is controlled by a complex set of regulatory mechanisms
which are still not fully understood. Despite this, misregulation of Hox gene expression
can lead to severe developmental abnormalities and various forms of disease.
This work addresses the way in which small non-coding RNAs (microRNAs, miRNAs)
regulate Hox gene expression and function during development. To do this we use the
Drosophila Hox gene Ultrabithorax (Ubx) as a paradigm for Hox gene function.
Using a suite of genetic methods we first uncover a novel regulatory interaction
between Drosophila Ubx and the miR-310C family of miRNAs during the development
of the haltere, a small dorsal appendage involved in flight control. We also show that
this miRNA cluster is required to fine tune Ubx expression. Furthermore, our data
provides insight into the role played by Ubx during appendage development.
Secondly, using a next generation RNA sequencing approach, we identify the full
repertoire of miRNAs present in two serially homologous appendages of Drosophila –
the wing and haltere. Our results show that these morphologically distinct appendages
have divergent miRNA profiles, including miRNAs which display appendage-specific
expression patterns. In addition, combining these profiles with available transcriptomic
data enabled us to study how miRNAs are integrated into the Ubx gene regulatory
networks that govern haltere development. This analysis suggests that haltere miRNAs
reinforce the regulatory programmes installed by Ubx during haltere development.
Our work therefore contributes to the understanding of the regulatory function of
miRNAs during development and sheds light on the ways in which Hox gene
expression can contribute to the formation of complex morphological structures.

With 9 million people in the UK alone suffering from deafness or hearing impairment, there is a concerted effort to develop effective treatments. To address this, one strategy is to recruit mechanisms naturally occurring during inner ear development, but this first requires a clear picture of the normal molecular mechanisms underlying this developmental process.

Fibroblast growth factors (FGFs) are short-range extracellular signalling molecules, with Fgf3 and Fgf10 already shown to be critical for the earliest event of inner ear induction. Interestingly these ligands are also expressed in the inner ear itself, and mutations in both Fgf3 and Fgf10 have independently been linked to sensorineural deafness in humans. This project is focussed on unravelling the molecular mechanisms controlling their expression in the inner ear.

Bioinformatic analysis of the Fgf3 and Fgf10 enhancer regions revealed the presences of putative binding sites for retinoic acid (RA). RA is a key signalling molecule in inner ear development with both excess and deficit leading to inner ear abnormalities. First, a novel, non-invasive method of RA administration via sugar pellet was tested and proved to be efficient and reliable alternative to gavage. Using an Fgf3-lacZ reporter mouse, the effects of RA excess on Fgf3 expression were investigated in detail. In addition, preliminary studies of the effects of RA on Fgf10 were also carried out. Both Fgf3 and Fgf10 were downregulated with high doses of RA, confirming previous in vitro studies. In addition, detailed analysis of Fgf3-lacZ embryos exposed to RA revealed that downregulation critically depends on the dose and time of administration.

To further explore other direct regulators of Fgf10 expression, additional reporter constructs were also generated for functional analysis in mouse and in chick. To date, analysis of electroporated chick embryos shows that ear specific Fgf10 regulation may not be conserved between two species.

Aim
Habitat loss continues to cause loss of biodiversity. To quantify the effects of land-use change on the diversity and composition of ecological communities – in terms of functional groups – we make modelled estimates of the impact of past and future (to 2050) land-use change on the overall diversity and dietary guild structure of tropical forest bird communities.

Location
Tropical and subtropical forests (40° S to 40° N).

Methods
Using a likelihood-based model, we project the impact of land-use intensity on the diversity and functional structure of tropical forest bird communities, including an estimation of uncertainty. To explore the extent to which predicted impacts are determined by the inherent sensitivity of communities because of the traits possessed by the species present, we quantify communities in terms of trait composition and explore relationships between trait composition and diversity/guild loss.

Results
We estimate that habitat loss has led to an average decrease of 4% in total abundance, but with marked differences across different guilds, leading to substantial changes in community composition: an 11.4% loss of frugivores, 7.8% loss of nectarivores and 7.3% loss of insectivores, and a 4.0% gain of other herbivores. Projected land-use change is predicted to result in average future losses of 1% of total abundance, 1% of each of frugivores, nectarivores and insectivores, but no average change in the abundance of other herbivores. Past and future changes have varied substantially across the biome, owing to variation in land-use change and in the initial trait composition of communities.

Main conclusions
We predict that marked changes in community structure have occurred in the past and will occur in the future, with disproportionate losses of frugivores, nectarivores and insectivores compared with other species; these species are known to provide important ecosystem services. In the past, South America has been particularly strongly affected, while Southeast Asia will experience the strongest impacts in the future.

Anthropogenic activities are causing widespread degradation of ecosystems worldwide, threatening the ecosystem services upon which all human life depends. Improved understanding of this degradation is urgently needed to improve avoidance and mitigation measures. One tool to assist these efforts is predictive models of ecosystem structure and function that are mechanistic: based on fundamental ecological principles. Here we present the first mechanistic General Ecosystem Model (GEM) of ecosystem structure and function that is both global and applies in all terrestrial and marine environments. Functional forms and parameter values were derived from the theoretical and empirical literature where possible. Simulations of the fate of all organisms with body masses between 10 µg and 150,000 kg (a range of 14 orders of magnitude) across the globe led to emergent properties at individual (e.g., growth rate), community (e.g., biomass turnover rates), ecosystem (e.g., trophic pyramids), and macroecological scales (e.g., global patterns of trophic structure) that are in general agreement with current data and theory. These properties emerged from our encoding of the biology of, and interactions among, individual organisms without any direct constraints on the properties themselves. Our results indicate that ecologists have gathered sufficient information to begin to build realistic, global, and mechanistic models of ecosystems, capable of predicting a diverse range of ecosystem properties and their response to human pressures.

Social groups are at particular risk for parasite infection, which is heightened in eusocial insects by the low genetic diversity of individuals within a colony. To combat this, adult ants have evolved a suite of defenses to protect each other, including the production of antimicrobial secretions. However, it is the brood in a colony that are most vulnerable to parasites because their individual defenses are limited, and the nest material in which ants live is also likely to be prone to colonization by potential parasites. Here, we investigate in two ant species whether adult workers use their antimicrobial secretions not only to protect each other but also to sanitize the vulnerable brood and nest material. We find that, in both leaf-cutting ants and weaver ants, the survival of the brood was reduced and the sporulation of parasitic fungi from them increased, when the workers nursing them lacked functional antimicrobial-producing glands. This was the case for both larvae that were experimentally treated with a fungal parasite (Metarhizium) and control larvae which developed infections of an opportunistic fungal parasite (Aspergillus). Similarly, fungi were more likely to grow on the nest material of both ant species if the glands of attending workers were blocked. The results show that the defense of brood and sanitization of nest material are important functions of the antimicrobial secretions of adult ants and that ubiquitous, opportunistic fungi may be a more important driver of the evolution of these defenses than rarer, specialist parasites.

Alpha- and beta-tectorin (Tecta and Tectb) are major non-collagenous components of
the tectorial membrane (TM). The presence of a zona pellucida (ZP) domain in both
tectorins suggests that Tecta and Tectb can form hetero- or homopolymers. It is unclear,
however, how these proteins assemble to form the TM matrix. The mechanisms of
apical targeting, secretion and processing of the tectorins are also unexplored. I used
fluorescently-tagged tectorin constructs for stable transfection into polarised epithelial
MDCK cells or transient expression in mouse cochlear cultures to develop an in vitro
model of TM matrix assembly. Significant amounts of matrix were not observed with
stable tectorin expression in monolayer cultures of MDCK cells. In contrast, I observed
substantial amounts of dense extracellular matrix on the apical surfaces of outgrowth
zone cells when cochlear cultures were transiently transfected with either Tecta or
Tectb. When ectopically expressed in hair cells, Tecta and Tectb locate to the distal tips
of the hair bundle.

To study the role of the inner-ear protein Ceacam16 in hearing, we generated a
Ceacam16 functional null mouse model. The Ceacam16 gene was inactivated by
targeted replacement of exons 2-5 with the bacterial lacZ gene. β-gal staining I
performed reveals that Ceacam16 is expressed in the epithelial cells of the spiral limbus
and inner sulcus, and in both the pillar cells and Deiter’s cells. I first detected the presence of Ceacam16 in the TM at P12, four days before the defined striated-sheet
matrix is observed. Transmission electron microscopy reveals a complete loss of
striated-sheet matrix in Ceacam16 null mice in comparison to the wild-type.

The results of this thesis suggest neonatal mouse cochlear cultures as a model for
studying tectorin-based extracellular matrix production and also reveal that Ceacam16 is
required for normal formation and/or maintenance of striated-sheet matrix.

Small Open Reading Frames (smORFs) are short DNA sequences able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others showed previously that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport, and hence influencing regular muscle contraction, in the Drosophila heart. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, where they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.

Cooperative transport, defined as multiple individuals simultaneously moving an object, has arisen many times in ants, but is otherwise extremely rare in animals. Here we review the surprisingly sparse literature available on cooperative transport. Cooperative transport abilities in ants are a continuum, but three general syndromes are described: uncoordinated transport, in which transport is slow, poorly coordinated and characterised by frequent and long deadlocks; encircling coordinated transport, in which transport is fast, well coordinated, and with few deadlocks; and forward-facing coordinated transport, carried out exclusively by army ants, in which one worker, usually of larger size, straddles an item at the front while one or more smaller workers help to lift at the back. In the two coordinated syndromes, the groups of ants involved constitute teams, and specialised recruitment to large items and adjustment of carrier number to match item size may occur. Some features of cooperative transport are specific adaptations, whilst others are already present in the behaviour of ants carrying items alone. One major benefit of cooperative transport appears to be that it allows a colony to utilize large food items in an environment with aggressive or dominant competitors by quickly removing the item to the nest rather than having to cut it up or consume it on the spot. In addition, compared to individual transport, cooperative transport may have other benefits such as increased transport speed or efficiency.

The study of cooperative transport also includes computer simulations and robots. These provide biologists with new perspectives and also formalise questions for further study. Likewise, lessons learned from cooperative transport in ants can inform computer scientists and roboticists.

Histone H2AX is required to maintain genomic stability in cells and to suppress malignant transformation of lymphocytes in mice. H2ax(-/-)p53(-/-) mice succumb predominantly to immature alphabeta T-cell lymphomas with translocations, deletions, and genomic amplifications that do not involve T-cell receptor (TCR). In addition, H2ax(-/-)p53(-/-) mice also develop at lower frequencies B and T lymphomas with antigen receptor locus translocations. V(D)J recombination is initiated through the programmed induction of DNA double-strand breaks (DSBs) by the RAG1/RAG2 endonuclease. Because promiscuous RAG1/RAG2 cutting outside of antigen receptor loci can promote genomic instability, H2ax(-/-)p53(-/-) T-lineage lymphomas might arise, at least in part, through erroneous V(D)J recombination. Here, we show that H2ax(-/-)p53(-/-)Rag2(-/-) mice exhibit a similar genetic predisposition as do H2ax(-/-)p53(-/-) mice to thymic lymphoma with translocations, deletions, and amplifications. We also found that H2ax(-/-)p53(-/-)Rag2(-/-) mice often develop thymic lymphomas with loss or deletion of the p53(+) locus. Our data show that aberrant V(D)J recombination is not required for rapid onset of H2ax/p53-deficient thymic lymphomas with genomic instability and that H2ax deficiency predisposes p53(-/-)Rag2(-/-) thymocytes to transformation associated with p53 inactivation. Thus, H2AX is essential for suppressing the transformation of developing thymocytes arising from the aberrant repair of spontaneous DSBs

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis

Histone H3 serine 10 phosphorylation is a hallmark of mitotic chromosomes, but its full function remains to be elucidated. We report here that two SR protein splicing factors, SRp20 and ASF/SF2, associate with interphase chromatin, are released from hyperphosphorylated mitotic chromosomes, but reassociate with chromatin late in M-phase. Inhibition of Aurora B kinase diminished histone H3 serine 10 phosphorylation and increased SRp20 and ASF/SF2 retention on mitotic chromosomes. Unexpectedly, we also found that HP1 proteins interact with ASF/SF2 in mitotic cells. Strikingly, siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on mitotic chromatin. Finally, ASF/SF2-depleted cells released from a mitotic block displayed delayed G0/G1 entry, suggesting a functional consequence of these interactions. These findings underscore the evolving role of histone H3 phosphorylation and demonstrate a direct, functional, and histone-modification-regulated association of SRp20 and ASF/SF2 with chromatin

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites.

Ataxia telangiectasia (A-T) is a disorder characterized by cerebellar degeneration, immunodeficiency, genomic instability and genetic predisposition to lymphoid malignancies with translocations involving antigen receptor loci. The Ataxia Telangiectasia Mutated gene encodes the ATM kinase, a central transducer of DNA damage signals. Until recently, the etiology of the lymphoid phenotype in A-T patients and the mechanisms by which ATM ensures normal repair of DNA double strand break (DSB) intermediates during antigen receptor diversification reactions remained poorly understood. Last year, Bredemeyer et al. (Nature 2006; 442:466-70) demonstrated that ATM stabilizes chromosomal V(D)J recombination DSB intermediates, facilitates DNA end joining and prevents broken DNA ends from participating in chromosome deletions, inversions and translocations. A more recent study by Callen et al. (Cell 2007; 130:63-75) highlighted the importance of ATM-mediated checkpoints in blocking the long-term persistence and transmission of un-repaired DSBs in developing lymphocytes. Collectively, these results have provided complementary mechanistic insights into ATM functions in V(D)J recombination that can account for the lymphoid tumor-prone phenotype associated with A-T

Epstein-Barr virus (EBV) immortalises resting B-lymphocyctes and is associated with a
diverse range of cancers and establishes a persistent, latent infection in >90% of the
world-wide population. Epstein-Barr virus nuclear antigen (EBNA) 3C is one of only
six EBV latent proteins that are crucial for B-cell transformation. EBNA3C is known to
disrupt cell-cycle control and to progress phase transition at G1/S and G2/M under
conditions where cells should growth arrest, but the mechanism by which EBNA3C
does this has not been fully determined. The cell-cycle regulator response gene to
complement (RGC) 32 was found to be upregulated in EBNA3C-expressing cells in
microarray experiments carried out previously. RGC-32 is involved in cell-cycle
activation and also plays a role in G1/S and G2/M transition. I have shown that both
EBNA3C-expressing cell-lines with upregulated RGC-32 and cell-lines overexpressing
RGC-32 alone displayed disrupted G2/M checkpoint control indicating that EBNA3C
may overcome cell-cycle control by upregulation of RGC-32. I also confirmed that
RGC-32 increases the in vitro kinase activity of CDK1, the key mitotic kinase essential
for G2/M transition. Surprisingly, my data showed that EBNA3C only activated RGC-
32 transcription in reporter assays at a very low-level, but stabilised the RGC-32
mRNA. Further studies investigating the differential expression of RGC-32 in EBVpositive
and negative cells demonstrated that RGC-32 is upregulated in LCLs and
tumour (Burkitt’s lymphoma) cell-lines expressing the full panel of latent genes, but
intriguingly highly expressed in Burkitt’s lymphoma cell-lines expressing only EBNA
1. I found that this expression pattern correlated with expression of the RUNX1
transcription factor. Reporter assays revealed that RUNX1 was able to activate the
RGC-32 promoter. Together, this data indicates a new mechanism by which EBNA 3C
can disrupt the G2/M checkpoint and highlights a link between RUNX1 and RGC-32
expression in B-cells.

Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCR) and copy number variations (CNV). GCRs/CNVs underlie human genomic disorders1 and are a feature of cancer2. During cancer development environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs/CNVs are proposed to contribute to cancer development and therapy resistance3. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination (HR) or microhomology-primed re-initiation (FoSTeS/MMBIR)4,5. Here we ascertain the consequences of replication with a fork restarted by HR. We identify a new mechanism of chromosomal rearrangement: recombination-restarted forks have an exceptionally high propensity to execute a U-turn at small inverted repeats (up to 1:40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer and to non-recurrent CNVs in genomic disorders

In migrating NRK cells, aPKCs control the dynamics of turnover of paxillin-containing focal adhesions (FA) determining migration rate. Using a proteomic approach (two-dimensional fluorescence difference gel electrophoresis), dynein intermediate chain 2 (dynein IC2) was identified as a protein that is phosphorylated inducibly during cell migration in a PKC-regulated manner. By gene silencing and co-immunoprecipitation studies, we show that dynein IC2 regulates the speed of cell migration through its interaction with paxillin. This interaction is controlled by serine 84 phosphorylation, which lies on the aPKC pathway. The evidence presented thus links aPKC control of migration to the dynein control of FA turnover through paxillin

World governments have committed to halting human-induced extinctions and safeguarding important sites for biodiversity by 2020, but the financial costs of meeting these targets are largely unknown. We estimate the cost of reducing the extinction risk of all globally threatened bird species (by ≥1 IUCN Red List category) to be US$0.875-1.23 billion annually over the next decade, of which 12% is currently funded. Incorporating threatened non-avian species increases this total to US$3.41-$4.76 billion annually. We estimate that protecting and effectively managing all terrestrial sites of global avian conservation significance (11,731 Important Bird Areas) would cost US$65.1 billion annually. Adding sites for other taxa increases this to US$76.1 billion annually. Meeting these targets will require conservation funding to increase by at least an order of magnitude.

A key element in the efficient allocation of scarce resources for conservation is the identification of areas of high biological value and high threat. Habitat loss and human population density have proved useful predictors of spatial variation in the current threat status of species albeit at coarse spatial resolution. We present a global analysis, intersecting Endemic Bird Areas (EBAs), to which restricted-range bird species are endemic, with fine-scale data of agricultural extent and human population density and test: (a) how well variation in land use mapped at 0.5degrees x 0.5degrees resolution predicts spatial variation in threat status of species and (b) how the predictive power compares with that of human population density mapped at the same resolution. Variation among EBAs in the proportion of restricted-range species that are threatened can be predicted by both the proportion of land used for agriculture and human population density. Agricultural land use was a better predictor of threat status than human population. Further, the average levels of threat attributable to agriculture were better predicted by land use than human population density, whereas threats due to causes other than agriculture were equally well predicted by land use and human population density. We fitted quantitative empirical models to describe these relationships. These results could be used, together with spatially explicit future scenarios of land use change, to project the geographical distribution and magnitude of future threats to birds at global and regional scales.

Energetically costly behaviours, such as flight, push physiological systems to their limits requiring metabolic rates (MR) that are highly elevated above the resting MR (RMR). Both RMR and MR during exercise (e.g. flight or running) in birds and mammals scale allometrically, although there is little consensus about the underlying mechanisms or the scaling relationships themselves. Even less is known about the allometric scaling of RMR and MR during exercise in insects. We analysed data on the resting and flight MR (FMR) of over 50 insect species that fly to determine whether RMR and FMR scale allometrically. RMR scaled with body mass to the power of 0.66 (M-0.66), whereas FMR scaled with M-1.10. Further analysis suggested that FMR scaled with two separate relationships; insects weighing less than 10 mg had fourfold lower FMR than predicted from the scaling of FMR in insects weighing more than 10 mg, although both groups scaled with M-0.86. The scaling exponents of RMR and FMR in insects were not significantly different from those of birds and mammals, suggesting that they might be determined by similar factors. We argue that low FMR in small insects suggests these insects may be making considerable energy savings during flight, which could be extremely important for the physiology and evolution of insect flight

Our aim in conducting annual horizon scans is to identify issues that, although currently receiving little attention, may be of increasing importance to the conservation of biological diversity in the future. The 15 issues presented here were identified by a diverse team of 22 experts in horizon scanning, and conservation science and its application. Methods for identifying and refining issues were the same as in two previous annual scans and are widely transferable to other disciplines. The issues highlight potential changes in climate, technology and human behaviour. Examples include warming of the deep sea, increased cultivation of perennial grains, burning of Arctic tundra, and the development of nuclear batteries and hydrokinetic in-stream turbines

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A)(+) RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G(2) phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle

We consider approaches to brain dynamics and function that have been claimed to be Darwinian. These include Edelman’s theory of neuronal group selection, Changeux’s theory of synaptic selection and selective stabilization of pre-representations, Seung’s Darwinian synapse, Loewenstein’s synaptic melioration, Adam’s selfish synapse and Calvin’s replicating activity patterns. Except for the last two, the proposed mechanisms are selectionist but not truly Darwinian, because no replicators with information transfer to copies and hereditary variation can be identified in them. All of them fit, however, a generalized selectionist framework conforming to the picture of Price’s covariance formulation, which deliberately was not specific even to selection in biology, and therefore does not imply an algorithmic picture of biological evolution. Bayesian models and reinforcement learning are formally in agreement with selection dynamics. A classification of search algorithms is shown to include Darwinian replicators (evolutionary units with multiplication, heredity and variability) as the most powerful mechanism for search in a sparsely occupied search space. Examples are given of cases where parallel competitive search with information transfer among the units is more efficient than search without information transfer between units. Finally, we review our recent attempts to construct and analyze simple models of true Darwinian evolutionary units in the brain in terms of connectivity and activity copying of neuronal groups. Although none of the proposed neuronal replicators include miraculous mechanisms, their identification remains a challenge but also a great promise.

Poly(ADP-ribose) polymerase 1 (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long, branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodeling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.

CNS axons differ in diameter (d) by nearly 100-fold (∼0.1-10 μm); therefore, they differ in cross-sectional area (d(2)) and volume by nearly 10,000-fold. If, as found for optic nerve, mitochondrial volume fraction is constant with axon diameter, energy capacity would rise with axon volume, also as d(2). We asked, given constraints on space and energy, what functional requirements set an axon's diameter? Surveying 16 fiber groups spanning nearly the full range of diameters in five species (guinea pig, rat, monkey, locust, octopus), we found the following: (1) thin axons are most numerous; (2) mean firing frequencies, estimated for nine of the identified axon classes, are low for thin fibers and high for thick ones, ranging from ∼1 to >100 Hz; (3) a tract's distribution of fiber diameters, whether narrow or broad, and whether symmetric or skewed, reflects heterogeneity of information rates conveyed by its individual fibers; and (4) mitochondrial volume/axon length rises ≥d(2). To explain the pressure toward thin diameters, we note an established law of diminishing returns: an axon, to double its information rate, must more than double its firing rate. Since diameter is apparently linear with firing rate, doubling information rate would more than quadruple an axon's volume and energy use. Thicker axons may be needed to encode features that cannot be efficiently decoded if their information is spread over several low-rate channels. Thus, information rate may be the main variable that sets axon caliber, with axons constrained to deliver information at the lowest acceptable rate.

The emergence of a unified cognitive behaviour relies on the coordination of specialized components that distribute across a ‘brain’, body and environment. Although a general dynamical mechanism involved in agent–environment integration is still largely unknown for behavioural robustness, discussions here are focussed on one of the most plausible candidate: the formation of distributed mechanisms working in transient during agent–environment coupling. This article provides discussions on this sort of coordination based on a mobile object-tracking task with situated, embodied and minimal agents, and tests for robust yet adaptive behaviour. The proposed scenario provides examples of behavioural mechanisms that counterbalance the functional organization of internal control activity and agents’ situatedness to enable the evolution of a two-agent interaction task. Discussions in this article suggest that future studies of distributed cognition should take into account that there are at least two possible modes of interpreting distributed mechanisms and that these have a qualitatively different effect on behavioural robustness.

Theoretical discussions and computational models of bio-inspired embodied and situated agents are introduced in this article capturing in simplified form the dynamical essence of robust, yet adaptive behavior. This article analyzes the general problem of how the dynamical coupling between internal control (brain), body and environment is used in the generation of specific behaviors. Based on the Evolutionary Robotics (ER) paradigm, four computational models are described to support discussions including descriptions on performance after a series of structural, sensorimotor or mutational perturbations, or are developed in the absence of them. Experimental results suggest that ‘dynamic determinacy’ – i.e. the continuous presence of a unique dynamical attractor that must be chased during functional behaviors – is a common dynamic phenomenon in the analyzed robust and adaptive agents. These agents show dynamical states that are definitely and unequivocally characterized via transient dynamics toward a unique, yet moving attractor at neural level for coherent actions. This determinacy emerges as a control strategy rooted on behavioral couplings and relies on mechanisms that are distributed on brain, body and environment. Different ways to induce further distribution of behavioral mechanisms are also discussed in this paper from a bio-inspired ER perspective.

This article investigates the emergence of robust behaviour in agents with dynamically limited controllers (monostable agents), and compares their performance to less limited ones (bistable agents). ‘Dynamically limited’ here refers to a reduced quantity of steady states that an agent controller exhibits when it does not receive stimulus from the environment. Agents are evolved for categorical perception, a minimal cognitive task, and must correlate approaching or avoiding movements based on (two) different types of objects. Results indicate a significant tendency to better behaviouralrobustness by monostable in contrast to bistable agents in the presence of sensorimotor, mutational, and structural perturbations. Discussions here focus on a further dependence to coupled dynamics by the former agents to explain such a tendency.

Over the last decade or so, sophisticated technological advances in array-based genomics have firmly established the contribution of structural alterations in the human genome to a variety of complex developmental disorders, and also to diseases such as cancer. In fact, multiple ‘novel’ disorders have been identified as a direct consequence of these advances. Our understanding of the molecular events leading to the generation of these structural alterations is also expanding. Many of the models proposed to explain these complex rearrangements involve DNA breakage and the coordinated action of DNA replication, repair and recombination machinery. Here, and within the context of Genomic Disorders, we will briefly overview the principal models currently invoked to explain these chromosomal rearrangements, including Non-Allelic Homologous Recombination (NAHR), Fork Stalling Template Switching (FoSTeS), Microhomology Mediated Break-Induced Repair (MMBIR) and Breakage–fusion–bridge cycle (BFB). We will also discuss an unanticipated consequence of certain copy number variations (CNVs) whereby the CNVs potentially compromise fundamental processes controlling genomic stability including DNA replication and the DNA damage response. We will illustrate these using specific examples including Genomic Disorders (DiGeorge/Veleocardiofacial syndrome, HSA21 segmental aneuploidy and rec (3) syndrome) and cell-based model systems. Finally, we will review some of the recent exciting developments surrounding specific CNVs and their contribution to cancer development as well as the latest model for cancer genome rearrangement; ‘chromothripsis’.

We investigated the effect of methionine sulfoximine (MetSox), a potent inhibitor of glutamine synthetase, on Mycobacterium tuberculosis. M. tuberculosis encodes four glutamine synthetases, of which MetSox targets the type I enzyme encoded by glnA1. Trancriptional profiling revealed that glutamate synthetase (gltB) and a type II glutamine synthetase (glnA3) were induced after exposure to MetSox. In addition, we observed a high rate (10−5) of spontaneous resistance to MetSox. All resistant strains had a single-nucleotide deletion in the 5’ region of glnA1, and Western analysis revealed that GlnA1 expression was increased in resistant as compared with sensitive strains. These data show that M. tuberculosis can respond to the effect of MetSox inhibition either by up-regulation of GlnA3 or by GlnA1. The high frequency of resistance suggests that MetSox and other compounds specifically targeting GlnA1 are not likely to become successful anti-mycobacterial agents.

Background: Staphylococcus aureus bacteremia is a common and serious infection worldwide and although treatment guidelines exist, there is little consensus on optimal management. In this study we assessed the variation in management and adherence to treatment guidelines of S. aureus bacteremia.

Methodology/Principal Findings: We prospectively recorded baseline clinical characteristics, management, and in-hospital outcome of all adults with S. aureus bacteremia treated consecutively over one year in eight centres in the United Kingdom, three in Vietnam and one in Nepal. 630 adults were treated for S. aureus bacteremia: 549 in the UK (21% methicillin-resistant), 80 in Vietnam (19% methicillin-resistant) and 1 in Nepal. In the UK, 41% had a removable infection focus (50% intravenous catheter-related), compared to 12% in Vietnam. Significantly (p<0.001) higher proportions of UK than Vietnamese patients had an echocardiogram (50% versus 28%), received more than 14 days antibiotic therapy (84% versus 44%), and received >50% of treatment with oral antibiotics alone (25% versus 4%). UK centres varied significantly (p<0.01) in the proportions given oral treatment alone for >50% of treatment (range 12-40%), in those treated for longer than 28 days (range 13-54%), and in those given combination therapy (range 14-94%). 24% died during admission: older age, time in hospital before bacteremia, and an unidentified infection focus were independent predictors of in-hospital death (p<0.001).

Conclusions/Significance: The management of S. aureus bacteremia varies widely between the UK and Vietnam and between centres in the UK with little adherence to published guidelines. Controlled trials defining optimal therapy are urgently required.

Over the last century, we have gone from ignorance as to why some diseases run in families to the availability of simple genetic tests that can be bought on the internet. And, from announcements of the death of Darwinism to the triumph of the modern theory of evolution. All this is thanks to the fruit fly, the guinea pig, the zebra fish and a handful of other organisms, which have helped us unravel one of life's greatest mysteries - inheritance. Jim Endersby's strikingly original book tells the history of modern biology through the stories of the animals and plants that made it possible, showing how the guinea-pig and its colleagues have played a pivotal role in our gradual understanding of what genes are and what they do. By spending years laboriously breeding these animals and plants, observing the consequences and extrapolating - sometimes quite wildly - from these observations, scientists have gradually come to understand how inheritance shapes generations to come. In telling their stories, Endersby reveals the development of perhaps the most significant science of our times. Endersby traces his story from Darwin hand-pollinating passionflowers in his back-garden in an effort to find out whether his decision to marry his cousin had harmed their children, to today's high-tech laboratories, full of shoals of shimmering zebra fish, whose bodies are transparent until they are mature, allowing scientists to watch every step as a single fertilised cell multiplies to become the millions of specialised cells that make up a new fish. Each story has - piece by piece - revealed how DNA determines the characteristics of the adult organism. Not every organism was as cooperative as the fruit fly or zebra fish, some provided scientists with misleading answers or encouraged them to ask the wrong questions. Entertaining, surprising and enlightening by turns, this unusual and original view of the science of life also challenges us to consider the ethical dilemmas that biology presents today - when we have the capacity as never before to change the very nature of living things.

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

The development of limb cartilage involves complex signalling pathways allowing the formation of distinct segments of cartilage that are maintained in the fully developed joint. In this study, we investigated the Notch signalling pathway and its role in cartilage development. The differential distribution of the Notch signalling family of receptors and their corresponding ligands in developing avian (gallus gallus) cartilage revealed expression of Notch 1, Delta 1, Jagged 1 and Jagged 2 in all limb mesenchyme cells at the early stages of cartilage anlagen development, which were subsequently restricted to the developing cartilage element. Expression of both Notch 1 and Jagged 1 became increasingly restricted to the surface cartilage once joint cavity formation had occurred. Delta 1 and Jagged 1 were restricted to a layer of cells underneath the surface cartilage and were also observed in the hypertrophic chondrocytes, where Notch 1 expression was evident in stage 40-44 limbs. Notch 2, Notch 3 and Notch 4 were not evident in early stage limbs but were present after cavitation, although expression was lost in late stage limbs (stage 40-44). We also demonstrated that inhibition of the Notch pathway leads to altered Notch receptor expression, disrupting cartilage differentiation. From these data it is clear that Notch signalling is a necessary and critical factor in regulating cell fate decisions allowing controlled chondrogenesis, elongation and subsequent maintenance of limb cartilage.

Stented coronary angioplasty is the procedure of choice to re-establish patency in obstructed coronary arteries. However, the stent implantation procedure often leads to in-stent restenosis, a process that is characterized by stent strut colonization by macrophages and smooth muscle cells and by neointima formation. The present in vitro study investigates the effect of stent materials on the phenotypical features of monocyte/macrophages. Human peripheral blood monocytes from healthy donors (n = 7) were cultured up to 7 days on substrates mimicking: (i) the stent surface (i.e., electropolished stainless steel), (ii) the de-endothelialized vessel wall (collagen-based extracellular matrix gel), and (iii) thrombus (i.e., fibrin gel). The cells were analyzed by immunocytochemistry for their ability to express alpha-actin, a typical myofibroblast marker, by ELISA to determine PDGF-BB and TGF-beta1 secretion and by PCR to evaluate hyaluronan synthase 1, 2, and 3 genes expression. Data were statistically analyzed by ANOVA (Dunnett's test) and data considered significantly different at p </= 0.05. The data demonstrated that mononuclear cells adhering to stainless steel acquire a phenotype capable of expressing alpha-actin while secreting significantly higher levels of PDGF-BB and TGF-beta. The expression of the three hyaluronan synthase isoforms was also altered by the metal substrate, where cells expressed genes only for the isoforms synthesizing high molecular weight hyaluronan. This study therefore suggests that mononuclear cells adhering on the stent metal surface undergo phenotypical transformation into myofibroblast-like cells that are able to contribute to neointimal tissue synthesis.

Objective. Polo-like kinase 1 (Plk1) is a regulator of the cell cycle that has been implicated in the pathology of many cancers. We have investigated whether this kinase plays a role in multiple myeloma (MM) using the Plk1 inhibitor BI 2536.

Materials and Methods. We have used six MM cell lines and six patient-derived samples to determine the effects of the Plk1 inhibitor, BI 2536, on cell viability, apoptosis, and cytokinesis. We have also examined the effect of the microenvironment on these parameters and the effects of BI 2536 in combination with other antimyeloma agents.

Results. We show that MM cell lines and patient samples express PLK1 and that cell death by apoptosis occurs when Plk1 is inhibited. Cells treated with BI 2536 accumulate in the G(2)/M phase of the cell cycle causing endoduplication. The effects of BI 2536 are not abrogated when cells are cultured on extracellular matrix components, in the presence of interleukin-6, or with bone marrow stromal cells.

Conclusions. Plk1 inhibition leads to cell death in MM cell lines and patient myeloma samples. Our data suggest that inhibition of Plk1 may have potential use as a therapeutic strategy in multiple myeloma.

Galectin-1, the prototype of a family of beta-galactoside-binding proteins, has been implicated in a wide variety of biological processes. Data presented herein show that galectin-1 stimulates monocyte migration in a dose-dependent manner but is not chemotactic for macrophages. Galectin-1-induced monocyte chemotaxis is blocked by lactose and inhibited by an anti-galectin-1 antibody but not by nonspecific antibodies. Furthermore, galectin-1-mediated monocyte migration was significantly inhibited by MEK inhibitors in a rapid, time-dependent manner suggesting that MAP kinase pathways are involved in galectin-1. Migration was also almost completely blocked by pertussis toxin implying G-protein involvement in the galectin-1-induced chemotaxis. These results demonstrate a role for galectin-1 in monocyte chemotaxis which differs from galectin-3 in that macrophages are nonresponsive. Furthermore, our observations suggest that galectin-1 may be involved in chemoattraction at sites of inflammation in vivo and may contribute to disease processes such as atherosclerosis.

In-stent restenosis is a clinical complication following coronary angioplasty caused by the implantation of the metal device in the atherosclerotic vessel. Histological examination has shown a clear contribution of both inflammatory and smooth muscle cells (SMCs) to the deposition of an excess of neointimal tissue. However, the sequence of events leading to clinically relevant restenosis is unknown. This paper aims to study the phenotype of SMCs when adhering on substrates and exposed to biochemical stimuli typical of the early phases of stent implantation. In particular, human SMC phenotype was studied when adhering on extracellular matrix-like material (collagen-rich gel), thrombus-like material (fibrin gel) and stent material (stainless steel) in the presence or absence of a platelet-derived growth factor (PDGF) stimulus. Cells on the collagen and fibrin-rich substrates maintained their contractile phenotype. By contrast, cells on stainless steel acquired a secretory phenotype with a proliferation rate 50 per cent higher than cells on the natural substrates. Cells on stainless steel also showed an increase in PDGF-BB receptor expression, thus explaining the increase in proliferation observed when cells were subject to PDGF-BB stimuli. The stainless steel substrate also promoted a different pattern of β1-integrin localization and an altered expression of hyaluronan (HA) synthase isoforms where the synthesis of high-molecular-weight HA seemed to be favoured. These findings highlighted the induction of a phenotypic pattern in SMC by the stainless steel substrate whereby the formation of a HA-rich neointimal tissue is enhanced.

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity.

Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low.

Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.

A current challenge in neuroscience and systems biology is to better understand properties that allow organisms to exhibit and sustain appropriate behaviours despite the effects of perturbations (behavioural robustness). There are still significant theoretical difficulties in this endeavour, mainly due to the context-dependent nature of the problem. Biological robustness, in general, is considered in the literature as a property that emerges from the internal structure of organisms, rather than being a dynamical phenomenon involving agent-internal controls, the organism body, and the environment. Our hypothesis is that the capacity for behavioural robustness is rooted in dynamical processes that are distributed between agent ‘brain’, body, and environment, rather than warranted exclusively by organisms’ internal mechanisms. Distribution is operationally defined here based on perturbation analyses.

Evolutionary Robotics (ER) techniques are used here to construct four computational models to study behavioural robustness from a systemic perspective. Dynamical systems theory provides the conceptual framework for these investigations. The first model evolves situated agents in a goalseeking scenario in the presence of neural noise perturbations. Results suggest that evolution implicitly selects neural systems that are noise-resistant during coupling behaviour by concentrating search in regions of the fitness landscape that retain functionality for goal approaching. The second model evolves situated, dynamically limited agents exhibiting minimalcognitive behaviour (categorization task). Results indicate a small but significant tendency toward better performance under most types of perturbations by agents showing further cognitivebehavioural dependency on their environments. The third model evolves experience-dependent robust behaviour in embodied, one-legged walking agents. Evidence suggests that robustness is rooted in both internal and external dynamics, but robust motion emerges always from the systemin-coupling. The fourth model implements a historically dependent, mobile-object tracking task under sensorimotor perturbations. Results indicate two different modes of distribution, one in which inner controls necessarily depend on a set of specific environmental factors to exhibit behaviour, then these controls will be more vulnerable to perturbations on that set, and another for which these factors are equally sufficient for behaviours. Vulnerability to perturbations depends on the particular distribution.

In contrast to most existing approaches to the study of robustness, this thesis argues that behavioural robustness is better understood in the context of agent-environment dynamical couplings, not in terms of internal mechanisms. Such couplings, however, are not always the full determinants of robustness. Challenges and limitations of our approach are also identified for future studies.

Interactions between insect herbivores and host plants are fundamental, shaping both
ecosystem functions and community structure. One aspect of insect-plant interactions that
has received considerable attention recently is the indirect linkages between aboveground
and belowground insect herbivores via a shared host plant. To date, the relationship
between a maternal insect aboveground and her soil dwelling offspring has been largely
overlooked. This study aimed to examine the interactions between the adult insect and soil
dwelling larvae of the vine weevil (Otiorhynchus sulcatus) with reference to the preference–
performance hypothesis, using an agronomically important host plant, red raspberry (Rubus
ideaus).

A meta–analysis of aboveground–belowground insect herbivore interactions highlighted
that belowground Coleopteran herbivores positively impacted aboveground Homoptera
and that general predictions from conceptual models in the literature regarding the
direction of interactions between insects were correct, but not statistically significant. In
addition it was found that aboveground insect herbivores negatively influenced the survival
of belowground herbivores. The preference–performance hypothesis was not supported in
the findings for the vine weevil on raspberry. Instead a conflict between larval
development and adult egg laying behaviour was observed, with a significant reduction in
larval mass recorded when maternal adults fed on the same host plant. Larval mass was
decreased by 19% after prior conspecific root feeding, but maternal weevils did not
distinguish between plants with and without larvae for oviposition. Significant differences
between larval performance (abundances and mass) on the raspberry cultivars Glen Rosa
and Glen Ample were not correlated with adult oviposition aboveground. Instead, in some
instances, maternal egg laying was correlated with foliar nitrogen content, suggesting that
this may be an influential factor in the oviposition behaviour of adult vine weevils.
Significant differences seen in larval performance in the laboratory were not reflected in the
field, with adult vine weevil populations on Glen Rosa and Glen Ample showing no
significant differences in terms of abundance.

The findings from this study suggest that mother–offspring relationships in an
aboveground–belowground context warrant further consideration. In particular, the
identification of potential conflict between mother and offspring highlights another factor
influencing aboveground–belowground relationships that could consequently influence
terrestrial ecosystems.

An apoplastic pathway, the so-called bypass flow, is important for Na+ uptake in rice
under saline conditions. The primary aim of this thesis was to identify the point of entry
for bypass flow into rice roots subjected to salinity. Investigations using lateral rootless
mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara,
Nipponbare and Taichung 65, respectively) and seedlings of rice cv. IR36 showed that
the entry point, quantified using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid
(PTS), was not at the sites of lateral root emergence. However, PTS was identified in
the vascular tissue of lateral roots using both epifluorescence microscopy and confocal
laser scanning microscopy. Cryo-scanning electron microscopy and epifluorescence
microscopy of sections stained with berberine-aniline blue and Fluorol Yellow 088
revealed that an exodermis was absent in the lateral roots, suggesting that the lack of the
exodermis allowed PTS to pass through the cortical layers, enter the stele and be
transported to the shoot via the transpiration stream. These findings suggest a role for
the lateral roots of rice in bypass flow. The addition of polyethylene glycol (PEG) and
silicon (Si) to the culture solution significantly reduced Na+ uptake to the shoot by
reducing bypass flow through the lateral roots. PEG was found to be more effective than
Si. It was also shown that changing the relative humidity in the air around the shoots
had a significant effect on the magnitude of bypass flow and the flux of water across the
roots: the greater the flux of water through the roots, the greater the Na+ uptake and
bypass flow. Furthermore, results showed that recombinant inbred lines of rice with low
Na+ transport possessed low magnitudes of bypass flow, whereas lines with high Na+
transport had a high degree of bypass flow, indicating that bypass flow could be used as
a criterion for screening salt resistance in rice varieties.

The hemiparasite Rhinanthus minor is a common component of many northern temperate grasslands. It can have major impacts on ecosystem processes, and is often present at very high densities, therefore constituting an important potential food source for invertebrate herbivores. Thus, the aim of this thesis is to investigate the interaction between this hemiparasite and its invertebrate herbivores, and to explore the various ecological factors which are likely to affect this interaction.

In the first series of experiments the thesis explores how the density of the hemiparasite affects the composition of the vegetation, the performance of the hemiparasite and the levels of invertebrate herbivore damage it receives. The results of a field experiment and a greenhouse study demonstrated that hemiparasite density can adversely affect its own performance and survivorship and dramatically change the composition of the vegetation, but surprisingly appeared to have no impacts on the levels of herbivore damage the hemiparasite receives.

The second series of experiments investigated the impacts if host identity on the performance of the hemiparasite and how this affects its invertebrate herbivores. The results demonstrated that host identity can have a major impact on the performance of the hemiparasite and its herbivores; however, the indirect effects on the invertebrates appear to be species specific.

Thirdly, the thesis examines the effects of multiple host plants on the performance of R. minor and the knock-on effects for its invertebrate herbivores. Experiments
demonstrated that multiple hosts are beneficial to R. minor, and that the antiherbivore defensive properties conferred to the hemiparasite by certain host plants
are maintained in the presence of a second host species.

Finally, the impact of nutrient addition and host plant damage on the performance of the hemiparasite and on the performance of its invertebrate herbivores was examined. The experiments showed that while certain host plants have highly contrasting effects on the performance of the hemiparasite‟s herbivores, the addition of nutrients and impact of host plant damage largely remove these differences, while neither factor appeared to affect the performance of the hemiparasite.

The interaction between hosts and parasites in bird populations has been studied extensively. I use game theoretic methods to model this interaction. This has been done previously but has not been studied taking into account the detailed sequential nature of this game. I introduce models allowing the host and parasite to make a number of decisions which will depend on a number of natural factors. A sequence of events follows, which is broken down into two key stages; firstly the interaction between the host and the parasite adult, and secondly that between the host and the parasite chick. The final decision involves the host choosing whether to raise or abandon the chicks that are in the nest. There are certain natural parameters and probabilities which are central to these various decisions; in particular the host is generally uncertain whether parasitism has taken place, but can assess the likelihood of parasitism based upon certain cues (e.g. how many eggs remain in its nest).
I have taken elements of games which have been previously created and constructed my own models to fully describe this interaction. These parasites have different methods of parasitizing the nests of their hosts, and the hosts can in turn have different reactions to these parasites. This is later built into a model where there is more than one host nesting over a breeding season. We have a number of nesting sites and different time points in which the host can begin to nest. In the previous models the host was given the opportunity to abandon the nest. In this game the host is allowed to abandon and then restart the nesting process. The probability that the host is parasitized can be decided using a number of factors including the number of hosts laying during a given time period, the nesting site or the number of parasites during the course of the season.
Using these models we are able to find situations which match those which we have seen in nature. Also the models are able to predict what natural changes such as parasitism rate or mimicry will do to the interaction. Overall I believe these models to give as good an indication of the key elements of the interaction and how they can change over time.

The molecular mechanisms that control growth appear to be conserved across the animal kingdom, with nitric oxide regulation of cell proliferation and growth being found to be very significant. Indeed, in Drosophila larval development and mammalian systems NO has been shown to be particularly important in these processes, and previous work in our laboratory has identified the Drosophila forkhead transcription factor dFOXO as a critical target through which NO signalling exerts its regulatory effects on growth, although little is currently known concerning the precise mechanisms involved.

Accordingly, in this thesis, we investigate the processes through which NO may modulate growth and demonstrate that targeted expression of a constitutively active NO Synthase to whole larval salivary glands or clones of cells within the glands, results in reduced endoreplication and growth as measured by nuclear size. Targeted over expression of dFOXO itself is shown to result in similar phenotypes, and subsequent molecular analysis of potential signalling targets required for this inhibition of growth reveals that dFOXO, Thor and Myc expression are regulated in vivo by NO.

To elucidate if NO acts directly on dFOXO, the genetic interaction of components of the insulin signalling pathway is analysed, exploiting RNA interference to assay what components are necessary for the NO signal to be effectively transduced, and it is demonstrated that NO control of growth is not through sGC, one of the most significant known targets for NOmediated regulation in other organisms.

We subsequently investigated the roles of Thor, a Drosophila 4E-binding protein, and the kinase, Lk6, homologues of which are known to be important in growth regulation in other organisms, and thus potential effectors of NO and dFOXO. However our data demonstrated that neither Thor nor Lk6 are required for the inhibition of growth by NO.

Interestingly a potential anti-oncogenic effect of NO signalling was also revealed following analysis of interactions between NO and Ras or Myc induced growth in which NO was able reduce the overgrowth produce by both these oncogenes.

Overall this research confirms dFOXO as an essential target for NO induced inhibition of growth. The work also eliminates two dFOXO transcription targets, Thor and Lk6, as necessary for NO to regulate growth.

Biological systems involve features/behaviours of individuals and populations that are influenced by a multitude of factors. To explore the dynamics of such systems, a statistical description offers the possibility of testing hypotheses, drawing predictions and more generally, assessing our understanding.

In the work presented, I analyse the properties of various biological systems of two very different organisms: Pharaoh‟s ants (Monomorium pharaonis) and badgers (Meles
meles). The basis of the work, in the two projects on these biological systems, relies heavily on data collection and explaining observations using quantitative methods such
as statistical analysis and simulations.

In the first part of this thesis, I describe animal movement in space and time using data collected on the foraging behaviour of ants. A new model is presented which appears to reflect, with a high degree of accuracy, the behaviour of real organisms. This model constitutes the basis of the second chapter in which the qualities of searching strategies are explored in the context of optimal foraging. The final chapter of first part of this
thesis concludes with a detailed analysis of the rate of exploration of individuals. As an essential part of foraging, the rate of individuals leaving their nest is analysed using collected data, and contrasted with results derived from a mathematical model.

The second part of this thesis focuses on badgers. A first chapter explores the significance of palate maculation that is observed in badgers and relates their symmetry to parasitic infection. I then explore the population dynamics of a population of badgers subject to natural variation in climatic conditions. A first analysis is based on local climatic conditions, while a second analysis focuses on a more general property of climate (i.e. its unpredictability) to infer population dynamics.

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described [1], Rad60 has a Hus5-independent function.