G protein-coupled receptor kinase 2 (GRK2) is a Rho-activated scaffold protein for the ERK MAP kinase cascade

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs also regulate signalling downstream of other families of receptors and have a number of non-receptor substrates and binding partners. Here we identify RhoA_GTP and Raf1 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 is a RhoA effector that serves as a RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoA_GTP binding to the catalytic domain of the kinase. Exogenous GRK2 has previously been shown to increase ERK activation downstream of the epidermal growth factor receptor (EGFR). Here we find that GRK2-mediated ERK activation downstream of the EGFR is Rho-dependent and that treatment with EGF promotes RhoA_GTP binding and ERK scaffolding by GRK2. Depletion of GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced, Rho- and ERK-dependent thymidine incorporation in vascular smooth muscle cells (VSMCs). We therefore hypothesise that Rho-dependent ERK MAPK scaffolding by GRK2 downstream of the EGFR may have an important role in the vasculature, where increased levels of both GRK2 and RhoA have been associated with hypertension.     

Automated sperm head morphology analyzer for open-source software

Sperm head morphology has been identified as a characteristic that can be used to predict a male's semen quality. In the present study, we have developed an automated sperm head morphology analysis (ASMA) plug-in for open-source ImageJ software (http://rsbweb.nih.gov/ij/). We describe the plug-in's functionality, and confirm its validity for sperm head morphology analysis using fish sperm. Sperm head morphological measurements (length and width) made with the ASMA plug-in did not differ from manual measurements. Using the plug-in to measure sperm head-shaped objects of known size, the associated plug-in error rate was < 0.5%. Brightness and contrast ratios influenced sperm head measurements, suggesting the need for standardized protocols. This plug-in was effective at measuring elliptical (i.e., Atlantic cod) as well as slightly irregular (i.e., Chinook salmon) shaped sperm heads. In conclusion, our ASMA plug-in represents a versatile alternative to costly sperm morphology software.     

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics

Introduction

Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood.

Methods

We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy.

Results

We documented evidence for four major phenotypes of RA synovium – lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004).

Conclusions

These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119859","term_id":"NCT01119859"}}NCT01119859     

Age determination and growth of the pollan, Coregonus autumnalis pollan Thompson, of Lough Neagh, Northern Ireland

Scales from Lough Neagh pollan display a large number of checks, making age determination difficult. Sclerite counts showed that an annual check is formed on scales in May and a second accessory check in most young fish in October. The method of ageing from scales was supported by inspection of length-frequency plots and by following the growth of pollan in their first 2 years of life. The body-scale relationship was curvilinear. Back-calculation showed that pollan of both sexes attain a fork length of 29 cm in 5 years (1 ∞=28.9 cm; k = 0.65; l ₀= -0.06 year). There is no evidence that annual growth rates have changed since 1965. Possible environmental causes of scale check formation are discussed.     

The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration

Introduction  

Recent evidence suggests that intervertebral disc (IVD) cells derived from degenerative tissue are unable to respond to physiologically relevant mechanical stimuli in the 'normal' anabolic manner, but instead respond by increasing matrix catabolism. Understanding the nature of the biological processes which allow disc cells to sense and respond to mechanical stimuli (a process termed 'mechanotransduction') is important to ascertain whether these signalling pathways differ with disease. The aim here was to investigate the involvement of interleukin (IL)-1 and IL-4 in the response of annulus fibrosus (AF) cells derived from nondegenerative and degenerative tissue to cyclic tensile strain to determine whether cytokine involvement differed with IVD degeneration.     

An ecosystem evaluation framework for global seamount conservation and management

In the last twenty years, several global targets for protection of marine biodiversity have been adopted but have failed. The Convention on Biological Diversity (CBD) aims at preserving 10% of all the marine biomes by 2020. For achieving this goal, ecologically or biologically significant areas (EBSA) have to be identified in all biogeographic regions. However, the methodologies for identifying the best suitable areas are still to be agreed. Here, we propose a framework for applying the CBD criteria to locate potential ecologically or biologically significant seamount areas based on the best information currently available. The framework combines the likelihood of a seamount constituting an EBSA and its level of human impact and can be used at global, regional and local scales. This methodology allows the classification of individual seamounts into four major portfolio conservation categories which can help optimize management efforts toward the protection of the most suitable areas. The framework was tested against 1000 dummy seamounts and satisfactorily assigned seamounts to proper EBSA and threats categories. Additionally, the framework was applied to eight case study seamounts that were included in three out of four portfolio categories: areas highly likely to be identified as EBSA with high degree of threat; areas highly likely to be EBSA with low degree of threat; and areas with a low likelihood of being EBSA with high degree of threat. This framework will allow managers to identify seamount EBSAs and to prioritize their policies in terms of protecting undisturbed areas, disturbed areas for recovery of habitats and species, or both based on their management objectives. It also identifies seamount EBSAs and threats considering different ecological groups in both pelagic and benthic communities. Therefore, this framework may represent an important tool to mitigate seamount biodiversity loss and to achieve the 2020 CBD goals.     

The evolution of infertility: does hatching rate in birds coevolve with female polyandry?

Natural levels of infertility in many taxa are often remarkably high, amounting to a considerable fitness cost which one expects to be minimized by natural selection. Several mechanisms have been proposed as potential causes of infertility, including inbreeding depression, genetic incompatibilities and selfish genetic elements. Infertility may also be an inherent result of conflict over fertilization between the sexes in polyandrous species, either because too many sperm enter the egg or because of over‐efficient barriers to such polyspermic fertilizations. We generated phylogenetic independent contrasts to examine the variation in hatching success for a maximum of 58 species of birds in relation to two measures of female polyandry. Hatching success varied enormously across species (range: 61–100%), with a mean of 12% of eggs failing to hatch, but was not related to either the rate of extrapair paternity or to relative testes size. Thus, the causes of this significant fitness cost remains unclear and merits further examination by evolutionary biologists.     

Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms.

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.     

Atlantic Bluefin Tuna in the Gulf of Maine, I: Estimation of Seasonal Abundance Accounting for Movement, School and School-Aggregation Behaviour

Direct assessment of the abundance of highly migratory pelagic species, such as tuna, is rarely available and most indices are based on catch information. We estimate the seasonal abundance of North Atlantic bluefin tuna, Thunnus thynnus, in the Gulf of Maine (GOM) from a 3-year aerial survey conducted with commercial spotter pilots, while also utilizing findings from analyses of tracking and tagging data. We apply statistical correction and calibration to seasonal abundance estimates accounting for measured changes in horizontal and vertical movement behaviour, size, shape and aggregation of bluefin tuna schools. Our approach relies on ecological knowledge of bluefin tuna to extrapolate survey observations across areas not sampled by correcting survey abundance estimates based on range of movement search pattern and depth preference. We demonstrate how separate findings obtained through the analysis of data collected across different spatial and temporal scales can be integrated to correct and calibrate estimates of population abundance. We obtain fitted estimates of seasonal abundance of bluefin tuna in the GOM during 1994–1996 in the range of 45,000–51,000 individuals. If tuna behaviour is not accounted for, we estimate that the base or residual survey precision would be 4–7% determined from analysis of recent spotter survey data in the study region. We estimate the precision in estimating seasonal abundance accounting for tuna behaviour to lie within a range of 1,301–3,302%. Under hypothetical future improvements in survey design that achieve a precision of 20% in transect length and placement, we calculate net-precision to lie within a range of 82–93%. This calculation assumes reducible uncertainty in school size estimation and irreducible uncertainty in movement and school-aggregation behaviour. We infer that survey precision could be further reduced by 43–32% to attain 10–50% in which a 3–8 years adaptive survey design may reliably detect a seasonal abundance trend.     

Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensitivity

The maximal activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in quadriceps or soleus muscle from animals in which the sensitivity to insulin was changed. Most conditions caused no effect on the activities but exercise-training increased the activity of adenosine deaminase and cold exposure increased the activity of 5'-nucleotidase in soleus muscle: in addition, ageing decreased markedly the activities of all three enzymes in both muscles. When the activities are based on mg protein they are much higher in both white and brown adipose tissue than in muscle, suggesting that changes in adenosine concentration may be important in changing insulin sensitivity in adipose tissue whereas changes in adenosine receptor number may be more important in muscle.