Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure–function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H+-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses

Can the collective intentions of individual professionals within healthcare teams predict the team's performance : developing methods and theory

Background: Within implementation research, using theory-based approaches to understanding the behaviours of healthcare professionals and the quality of care that they reflect and designing interventions to change them is being promoted. However, such approaches lead to a new range of methodological and theoretical challenges pre-eminent among which are how to appropriately relate predictors of individual's behaviour to measures of the behaviour of healthcare professionals .The aim of this study was to explore the relationship between the theory of planned behaviour proximal predictors of behaviour (intention and perceived behavioural control, or PBC) and practice level behaviour. This was done in the context of two clinical behaviours – statin prescription and foot examination – in the management of patients with diabetes mellitus in primary care. Scores for the predictor variables were aggregated over healthcare professionals using four methods: simple mean of all primary care team members' intention scores; highest intention score combined with PBC of the highest intender in the team; highest intention score combined with the highest PBC score in the team; the scores (on both constructs) of the team member identified as having primary responsibility for the clinical behaviour. Methods: Scores on theory-based cognitive variables were collected by postal questionnaire survey from a sample of primary care doctors and nurses from northeast England and the Netherlands. Data on two clinical behaviours were patient reported, and collected by postal questionnaire survey. Planned analyses explored the predictive value of various aggregations of intention and PBC in explaining variance in the behavioural data. Results: Across the two countries and two behaviours, responses were received from 37 to 78% of healthcare professionals in 57 to 93% practices; 51% (UK) and 69% (Netherlands) of patients surveyed responded. None of the aggregations of cognitions predicted statin prescription. The highest intention in the team (irrespective of PBC) was a significant predictor of foot examination Conclusion: These approaches to aggregating individually-administered measures may be a methodological advance of theoretical importance. Using simple means of individual-level measures to explain team-level behaviours is neither theoretically plausible nor empirically supported; the highest intention was both predictive and plausible. In studies aiming to understand the behaviours of teams of healthcare professionals in managing chronic diseases, some sort of aggregation of measures from individuals is necessary. This is not simply a methodological point, but a necessary step in advancing the theoretical and practical understanding of the processes that lead to implementation of clinical behaviours within healthcare teams

Fluorescence Resonance Energy Transfer (FRET) as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH) proteins

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy

Female economic dependence and the morality of promiscuity

This article is made available through the Brunel Open Access Publishing Fund. Copyright @ The Author(s) 2014.In environments in which female economic dependence on a male mate is higher, male parental investment is more essential. In such environments, therefore, both sexes should value paternity certainty more and thus object more to promiscuity (because promiscuity undermines paternity certainty). We tested this theory of anti-promiscuity morality in two studies (N = 656 and N = 4,626) using U.S. samples. In both, we examined whether opposition to promiscuity was higher among people who perceived greater female economic dependence in their social network. In Study 2, we also tested whether economic indicators of female economic dependence (e.g., female income, welfare availability) predicted anti-promiscuity morality at the state level. Results from both studies supported the proposed theory. At the individual level, perceived female economic dependence explained significant variance in anti-promiscuity morality, even after controlling for variance explained by age, sex, religiosity, political conservatism, and the anti-promiscuity views of geographical neighbors. At the state level, median female income was strongly negatively related to anti-promiscuity morality and this relationship was fully mediated by perceived female economic dependence. These results were consistent with the view that anti-promiscuity beliefs may function to promote paternity certainty in circumstances where male parental investment is particularly important

Getting into hot water:sick guppies frequent warmer thermal conditions

Ectotherms depend on the environmental temperature for thermoregulation and exploit thermal regimes that optimise physiological functioning. They may also frequent warmer conditions to up-regulate their immune response against parasite infection and/or impede parasite development. This adaptive response, known as ‘behavioural fever’, has been documented in various taxa including insects, reptiles and fish, but only in response to endoparasite infections. Here, a choice chamber experiment was used to investigate the thermal preferences of a tropical freshwater fish, the Trinidadian guppy (Poecilia reticulata), when infected with a common helminth ectoparasite Gyrodactylus turnbulli, in female-only and mixed-sex shoals. The temperature tolerance of G. turnbulli was also investigated by monitoring parasite population trajectories on guppies maintained at a continuous 18, 24 or 32 °C. Regardless of shoal composition, infected fish frequented the 32 °C choice chamber more often than when uninfected, significantly increasing their mean temperature preference. Parasites maintained continuously at 32 °C decreased to extinction within 3 days, whereas mean parasite abundance increased on hosts incubated at 18 and 24 °C. We show for the first time that gyrodactylid-infected fish have a preference for warmer waters and speculate that sick fish exploit the upper thermal tolerances of their parasites to self medicate

Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5′-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus

Bistability in Apoptosis by Receptor Clustering

Apoptosis is a highly regulated cell death mechanism involved in many physiological processes. A key component of extrinsically activated apoptosis is the death receptor Fas, which, on binding to its cognate ligand FasL, oligomerize to form the death-inducing signaling complex. Motivated by recent experimental data, we propose a mathematical model of death ligand-receptor dynamics where FasL acts as a clustering agent for Fas, which form locally stable signaling platforms through proximity-induced receptor interactions. Significantly, the model exhibits hysteresis, providing an upstream mechanism for bistability and robustness. At low receptor concentrations, the bistability is contingent on the trimerism of FasL. Moreover, irreversible bistability, representing a committed cell death decision, emerges at high concentrations, which may be achieved through receptor pre-association or localization onto membrane lipid rafts. Thus, our model provides a novel theory for these observed biological phenomena within the unified context of bistability. Importantly, as Fas interactions initiate the extrinsic apoptotic pathway, our model also suggests a mechanism by which cells may function as bistable life/death switches independently of any such dynamics in their downstream components. Our results highlight the role of death receptors in deciding cell fate and add to the signal processing capabilities attributed to receptor clustering.Comment: Accepted by PLoS Comput Bio

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from $\textit{Pyrococcus furiosus}$. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.Wellcome Trust Translational (Grant ID: 080083/Z/06/Z) and Seeding Drug Discovery Initiative (Grant ID: 91050/Z/10/Z) award