F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.     

Estimation of peak vertical velocity and relative load changes by subjective measures in weightlifting movements

To investigate the ability of the OMNI-RES (0–10) scale to estimate velocity and loading changes during sets to failure in the hang power clean (HPC) exercise. Eleven recreationally resistance-trained males (28.5 ± 3.5 years) with an average one-repetition maximum (1RM) value of 1.1 ± 0.07 kg body mass^-1 in HPC, were assessed on five separate days with 48 hours of rest between sessions. After determining the 1RM value, participants performed four sets to self-determined failure with the following relative loading ranges: 60% < 70%, 70 < 80%, 80 < 90% and > 90%. The peak vertical velocity (PVV), and Rating of Perceived Exertion (RPE) were measured for every repetition of each set. The RPE expressed after the first repetition (RPE-1) and when the highest value of PVV was achieved during the set (RPE-max) were similar and significantly lower than the RPE associated with a 5% (RPE-5%) and 10% (RPE-10%) drop in PVV. In addition, the RPE produced at failure was similar to RPE-5% only for the heaviest range (≥ 90%). Furthermore, RPE-1 was useful to distinguish loading zones between the four assessed ranges (60 < 70%, vs. 70 < 80%, vs. 80 < 90%, vs. ≥ 90%). The RPE seems to be useful to identify PVV changes (maximal, 5% and 10% drop) during continuous sets to self-determined failure and to distinguish 10% loading zone increments, from 60 to 100% of 1RM in the HPC exercise.     

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

Neuronal presynaptic terminals contain hundreds of neurotransmitter‐filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well‐known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5‐dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.     

Dopamine D2 receptors form higher order oligomers at physiological expression levels

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

A maximal isokinetic pedalling exercise for EMG normalization in cycling

An isometric maximal voluntary contraction (iMVC) is mostly used for the purpose of EMG normalization, a procedure described in the scientific literature in order to compare muscle activity among different muscles and subjects. However, the use of iMVC has certain limitations. The aims of the present study were therefore to propose a new method for the purpose of EMG amplitude normalization in cycling and assess its reliability. Twenty-three cyclists performed 10 trials of a maximal isokinetic protocol (MIP) on a cycle ergometer, then another four sub-maximal trials, whilst the EMG activity of four lower limbs muscles was registered. During the 10 trials power output (CV = 2.19) and EMG activity (CV between 4.46 and 8.70) were quite steady. Furthermore, their maximal values were reached within the 4th trial. In sub-maximal protocol EMG activity exhibited an increase as a function of exercise intensity.MIP entails a maximal dynamic contraction of the muscles involved in the pedalling action and the normalization session is performed under the same biomechanical conditions as the following test session. Thus, it is highly cycling-specific.MIP has good logical validity and within-subject reproducibility. Three trials are enough for the purpose of EMG normalization in cycling.     

Quantitative Trait Loci Controlling Vegetative Growth Rate in the Edible Basidiomycete Pleurotus ostreatus

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.     

Hydrogel-Forming Microneedles Prepared from “Super Swelling” Polymers Combined with Lyophilised Wafers for Transdermal Drug Delivery

We describe, for the first time, hydrogel-forming microneedle arrays prepared from “super swelling” polymeric compositions. We produced a microneedle formulation with enhanced swelling capabilities from aqueous blends containing 20% w/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na₂CO₃ and utilised a drug reservoir of a lyophilised wafer-like design. These microneedle-lyophilised wafer compositions were robust and effectively penetrated skin, swelling extensively, but being removed intact. In in vitro delivery experiments across excised neonatal porcine skin, approximately 44 mg of the model high dose small molecule drug ibuprofen sodium was delivered in 24 h, equating to 37% of the loading in the lyophilised reservoir. The super swelling microneedles delivered approximately 1.24 mg of the model protein ovalbumin over 24 h, equivalent to a delivery efficiency of approximately 49%. The integrated microneedle-lyophilised wafer delivery system produced a progressive increase in plasma concentrations of ibuprofen sodium in rats over 6 h, with a maximal concentration of approximately 179 µg/ml achieved in this time. The plasma concentration had fallen to 71±6.7 µg/ml by 24 h. Ovalbumin levels peaked in rat plasma after only 1 hour at 42.36±17.01 ng/ml. Ovalbumin plasma levels then remained almost constant up to 6 h, dropping somewhat at 24 h, when 23.61±4.84 ng/ml was detected. This work represents a significant advancement on conventional microneedle systems, which are presently only suitable for bolus delivery of very potent drugs and vaccines. Once fully developed, such technology may greatly expand the range of drugs that can be delivered transdermally, to the benefit of patients and industry. Accordingly, we are currently progressing towards clinical evaluations with a range of candidate molecules.     

Temporally unpredictable supplementary feeding may benefit endangered scavengers

The monitoring of an experimental feeding station established in northern Spain allowed the evaluation of how this type of resource, predictable in space but not in time, was exploited by a guild of avian scavengers in relation to factors such as season, hour of disposal and presence of the dominant species. The presence of Egyptian Vultures Neophron percnopterus at carcasses was more likely during spring, and richness and diversity of avian scavengers was lower during the summer and when Griffon Vultures Gyps fulvus arrived earlier. The temporal unpredictability of the resource may favour exploitation by smaller and less competitive scavengers. New European regulations may present an opportunity to develop effective conservation measures to support functional scavenger assemblages.