Effect of subunit on allosteric modulation of ion channel function in stably expressed human recombinant -aminobutyric acidA receptors determined using 36Cl ion flux.

ABSTRACT Inhibitory ␥-aminobutyric acid (GABA) A receptors are subject to modulation at a variety of allosteric sites, with pharmacology dependent on receptor subunit combination. The influence of different ␣ subunits in combination with ␤3␥2s was examined in stably expressed human recombinant GABA A receptors by measuring 36 Cl influx through the ion channel pore. Muscimol and GABA exhibited similar maximal efficacy at each receptor subtype, although muscimol was more potent, with responses blocked by picrotoxin and bicuculline. Receptors containing the ␣3 subunit exhibited slightly lower potency. The comparative pharmacology of a range of benzodiazepine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABA A receptors (␣1, ␣2, ␣3, ␣5), ␣5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at ␣3␤3␥2s with nonselective agonist chlordiazepoxide was greater than at ␣1, ␣2, or ␣5 (P Ͻ 0.001). The presence of an ␣4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower ␣4 affinity, and FG8205 displayed similar efficacy. Most striking were the lack of affinity/ efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 Ϯ 12%), CGS8216 (56 Ϯ 6%), CGS9895 (65 Ϯ 6%), and the weak partial inverse agonist Ro15-4513 (87 Ϯ 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5␣-pregnan-3␣-ol-20-one, but the type of ␣ subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at ␣4␤3␥2s (226 Ϯ 10% increase in the EC 20 response to GABA) than any other modulator. The rank order of potentiation for pregnanolone was ␣5 Ͼ ␣2 Ͼ ␣3 ϭ ␣4 Ͼ ␣1, for loreclezole ␣1 ϭ ␣2 ϭ ␣3 Ͼ ␣5 Ͼ ␣4, and for pentobarbital ␣4 ϭ ␣5 ϭ ␣2 Ͼ ␣1 ϭ ␣3

Automated PDF highlighting to support faster curation of literature for Parkinson's and Alzheimer's disease

Neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease are devastating and costly illnesses, a source of major global burden. In order to provide successful interventions for patients and reduce costs, both causes and pathological processes need to be understood. The ApiNATOMY project aims to contribute to our understanding of neurodegenerative disorders by manually curating and abstracting data from the vast body of literature amassed on these illnesses. As curation is labour-intensive, we aimed to speed up the process by automatically highlighting those parts of the PDF document of primary importance to the curator. Using techniques similar to those of summarisation, we developed an algorithm that relies on linguistic, semantic and spatial features. Employing this algorithm on a test set manually corrected for tool imprecision, we achieved a macro F1-measure of 0.51, which is an increase of 132% compared to the best bag-of-words baseline model. A user based evaluation was also conducted to assess the usefulness of the methodology on 40 unseen publications, which reveals that in 85% of cases all highlighted sentences are relevant to the curation task and in about 65% of the cases, the highlights are sufficient to support the knowledge curation task without needing to consult the full text. In conclusion, we believe that these are promising results for a step in automating the recognition of curation-relevant sentences. Refining our approach to pre-digest papers will lead to faster processing and cost reduction in the curation process

Evaluating operational AVHRR sea surface temperature data at the coastline using surfers

Sea surface temperature (SST) is an essential climate variable that can be measured routinely from Earth Observation (EO) with high temporal and spatial coverage. To evaluate its suitability for an application, it is critical to know the accuracy and precision (performance) of the EO SST data. This requires comparisons with co-located and concomitant in situ data. Owing to a relatively large network of in situ platforms there is a good understanding of the performance of EO SST data in the open ocean. However, at the coastline this performance is not well known, impeded by a lack of in situ data. Here, we used in situ SST measurements collected by a group of surfers over a three year period in the coastal waters of the UK and Ireland, to improve our understanding of the performance of EO SST data at the coastline. At two beaches near the city of Plymouth, UK, the in situ SST measurements collected by the surfers were compared with in situ SST collected from two autonomous buoys located ∼7 km and ∼33 km from the coastline, and showed good agreement, with discrepancies consistent with the spatial separation of the sites. The in situ SST measurements collected by the surfers around the coastline, and those collected offshore by the two autonomous buoys, were used to evaluate the performance of operational Advanced Very High Resolution Radiometer (AVHRR) EO SST data. Results indicate: (i) a significant reduction in the performance of AVHRR at retrieving SST at the coastline, with root mean square errors in the range of 1.0 to 2.0 °C depending on the temporal difference between match-ups, significantly higher than those at the two offshore stations (0.4 to 0.6 °C); (ii) a systematic negative bias in the AVHRR retrievals of approximately 1 °C at the coastline, not observed at the two offshore stations; and (iii) an increase in the root mean square error at the coastline when the temporal difference between match-ups exceeded three hours. Harnessing new solutions to improve in situ sampling coverage at the coastline, such as tagging surfers with sensors, can improve our understanding of the performance of EO SST data in coastal regions, helping inform users interested in EO SST products for coastal applications. Yet, validating EO SST products using in situ SST data at the coastline is challenged by difficulties reconciling the two measurements, which are provided at different spatial scales in a dynamic and complex environment

Future Ocean Observations to Connect Climate, Fisheries and Marine Ecosystems

Advances in ocean observing technologies and modeling provide the capacity to revolutionize the management of living marine resources. While traditional fisheries management approaches like single-species stock assessments are still common, a global effort is underway to adopt ecosystem-based fisheries management (EBFM) approaches. These approaches consider changes in the physical environment and interactions between ecosystem elements, including human uses, holistically. For example, integrated ecosystem assessments aim to synthesize a suite of observations (physical, biological, socioeconomic) and modeling platforms [ocean circulation models, ecological models, short-term forecasts, management strategy evaluations (MSEs)] to assess the current status and recent and future trends of ecosystem components. This information provides guidance for better management strategies. A common thread in EBFM approaches is the need for high-quality observations of ocean conditions, at scales that resolve critical physical-biological processes and are timely for management needs. Here we explore options for a future observing system that meets the needs of EBFM by (i) identifying observing needs for different user groups, (ii) reviewing relevant datasets and existing technologies, (iii) showcasing regional case studies, and (iv) recommending observational approaches required to implement EBFM. We recommend linking ocean observing within the context of Global Ocean Observing System (GOOS) and other regional ocean observing efforts with fisheries observations, new forecasting methods, and capacity development, in a comprehensive ocean observing framework

Seismic-frequency attenuation and moduli estimates using a fiber-optic strainmeter

Summary We have developed a fiber-optic strainmeter to estimate velocities and attenuation at seismic frequencies. The two main advantages of the new system compared to strain gage techniques are the higher sensitivity to deformations (moduli) and phase lags (attenuation), and that estimates are representative of bulk values. While stress-strain measurements using strain gages or ultrasonic wave propagation sample only part of the core sample, the fiber-optic strainmeter would analyze the rock sample response to an applied stress as a whole. Still, the system is under development and the first experiment on a Plexiglas sample showed that attenuation estimates are more robust than deformation estimates due to difficulties with light intensity. Initial rock measurements are made on a dolomite sample

Integrin-specific control of focal adhesion kinase and RhoA regulates membrane protrusion and invasion

Cell invasion through extracellular matrix (ECM) is a hallmark of the metastatic cascade. Cancer cells require adhesion to surrounding tissues for efficient migration to occur, which is mediated through the integrin family of receptors. Alterations in expression levels of b1 and b3 integrins have previously been reported in a number of human cancers. However, whether there are specific roles for these ubiquitous receptors in mediating cell invasion remains unclear. Here we demonstrate that loss of b1 but not b3 integrins leads to increased spread cell area and focal adhesion number in cells on 2D immobilized fibronectin. Increased adhesion numbers in b1 knockdown cells correlated with decreased cell migration on 2D surfaces. Conversely, cells depleted of b1 integrins showed increased migration speed on 3D cell-derived matrix as well as in 3D organotypic cultures and inverted invasion assays. This increased invasive potential was also seen in cells lacking b3 integrin but only in 3D cultures containing fibroblasts. Mechanistically, in situ analysis using FRET biosensors revealed that enhanced invasion in cells lacking b1 integrins was directly coupled with reduced activation of focal adhesion kinase (FAK) and the small GTPase RhoA resulting in formation of enhanced dynamic protrusions and increased invasion. These reductions in FAK-RhoA signal activationwere not detected in b3 knockdown cells under the same conditions. This data demonstrates a specific role for b1 integrins in the modulation of a FAK-RhoA-actomyosin signaling axis to regulate cell invasion throug

β1 and β3 integrins differentially contribute to RhoA activation during invasion.

<p>(A) Z-projections of >25 confocal z-stack images of specified cells expressing GFP-lifeact embedded in 3D ECM gels. Scale bar is 10 µm. Graphs show mean cell area and % of cell area occupied by membrane protrusions quantified from reconstructed confocal z-stack images of GFP-lifeact cells as shown. At least 35 cells quantified for each, error bars are SEM. * denotes p<0.01. (B) Example images and quantification of FRET analysis of RhoA activation in each cell type. Cells cultured in 3D gels either in presence or absence of human dermal fibroblasts (HDF). Bars show mean FRET efficiency (%) +/−SEM, n =  24 for each over 3 independent experiments. (D) Quantification of RhoA activation using analysis of RhoA FRET biosensor in control cells treated with control or integrin function blocking antibodies (left graph) or integrin knockdown cells plated in 3D gels in the presence of control media or conditioned media from human dermal fibroblasts (HDF). Bars are mean FRET efficiency +/−SEM, n = 30 cells over 3 independent experiments. * = p<0.01.</p

Fibroblast-dependent cell invasion is regulated by β1-dependent modulation of RhoA activity.

<p>(A) Example confocal images of cells plated in cell-derived matrices (CDM) and stained for phalloidin-Alexa488 (green) and (P)MLC-Alexa568 (red). Bottom panels show (P)MLC channel only. Scale bars are 10 µm. (B) Example confocal images of organotypic cultures stained with antibodies to (P)MLC (left panels). MDA MB 231 (GFP) cells are shown in right panels. Scale bars are 50 µm. (C) Example projected images of >15 confocal z-slices of control or knockdown cells expressing GFP-lifeact. Scale bars are 10 µm. (D) Quantification of protrusion area as a function of total cell area calculated from images as in (C). Bars represent mean % protrusion area per cell +/−SEM from 50 cells over 3 independent experiments. ** = p<0.01, * = p<0.005. (E) Quantification of invasion of shCon cells or β 1kd cells expressing ROCK or p190RhoGEF in organotypic assays in the absence of HDF (as in (B). Bars represent invasion index+/−SEM from 25 images over 2 independent experiments. ** = p<0.01, * = p<0.005.</p

Enhanced invasion in β1-silenced cells is regulated by attenuated FAK activity.

<p>(A) Western blot of lysates from specified cells either untreated or treated with 1 µM PF228 (FAK inhibitor) for 2 hours. Blot is probed for active (P-397) or total FAK. GAPDH serves as a loading control. Numbers below represent average active FAK levels as a % of control as quantified by densitometry from 4 independent experiments +/−SEM. (B) Western blot of lysates from shCon or β1kd cells treated with vehicle control or PF-228 at 100 nM (FAKi) and probed for P-FAK (Y-397) or total FAK. (C) Example images of shCon or β1kd cells expressing FAK FERM FRET biosensor embedded in 3D gels. Images in left panel show F-actin (phalloidin) and right panels show FRET efficiency heatmaps according to pseudocolour scale bar indicated. Graph shows quantification of >30 cells per specified condition. Bars represent mean FRET efficiency+/−SEM across 5 independent experiments. ** = p<0.01, * = p<0.005. (D) Quantification of protrusion area/cell of control or β1kd cells expressing GFP-lifeact and embedded in 3D gels. Cells were treated with DMSO or PF228 at 100 nM prior to analysis. Bars represent mean+/−SEM of 45 cells each over 2 experiments. * = p<0.01 (E) Quantification of invasion of specified cells into 3D gels treated with DMSO (vehicle control) or PF-228 at specified concentrations. Bars represent mean+/−SEM or 35 images across 3 independent experiments. ** = p<0.01, * = p<0.05.</p