Relative antagonism of mutants of the CGRP receptor extracellular loop 2 domain (ECL2) using a truncated competitive antagonist (CGRP8-37):evidence for the dual involvement of ECL2 in the two-domain binding model

The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach

The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.</jats:p

Absence of new psychoactive substances in wastewater from South Wales, UK, revealed by optimised liquid chromatography-time-of-flight analysis.

New psychoactive substances are produced and marketed to mimic the effects of their illicit counterparts and to attempt to evade drug tests and prosecution. Here, we present the optimisation, validation and application of an analytical method using liquid chromatography-time-of-flight mass spectrometry to detect and quantify 37 new psychoactive substances and illicit substances in wastewater from South Wales, UK, using a targeted analysis method. Sample preparation was performed using solid-phase extraction with Oasis HLB cartridges. The LC separation was performed using a YMC-Triart Phenyl 450 bar column (12 nm, 5 μm, 100 × 3 mm) which provided good separation and resolution for all targeted analytes with a run time of 9 min. The method was validated using the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recovery and matrix effects. The method was then applied to influent wastewater samples collected from two wastewater treatment plants in Wales, UK

A pyridinium derivative from Red Sea soft corals inhibited voltage-activated potassium conductances and increased excitability of rat cultured sensory neurones

BACKGROUND: Whole cell patch clamp recording and intracellular Ca(2+ )imaging were carried out on rat cultured dorsal root ganglion (DRG) neurones to characterize the actions of crude extracts and purified samples from Red Sea soft corals. The aim of the project was to identify compounds that would alter the excitability of DRG neurones. RESULTS: Crude extracts of Sarcophyton glaucum and Lobophyton crassum attenuated spike frequency adaptation causing DRG neurones to switch from firing single action potentials to multiple firing. The increase in excitability was associated with enhanced KCl-evoked Ca(2+ )influx. The mechanism of action of the natural products in the samples from the soft corals involved inhibition of voltage-activated K(+ )currents. An active component of the crude marine samples was identified as 3-carboxy-1-methyl pyridinium (trigonelline). Application of synthetic 3-carboxy-1-methyl pyridinium at high concentration (0.1 mM) also induced multiple firing and reduced voltage-activated K(+ )current. The changes in excitability of DRG neurones induced by 3-carboxy-1-methyl pyridinium suggest that this compound contributes to the bioactivity produced by the crude extracts from two soft corals. CONCLUSION: Sarcophyton glaucum and Lobophyton crassum contain natural products including 3-carboxy-1-methyl pyridinium that increase the excitability of DRG neurones. We speculate that in addition to developmental control and osmoregulation these compounds may contribute to chemical defenses

Perspective: Quantum Hamiltonians for optical interactions

The multipolar Hamiltonian of quantum electrodynamics is extensively employed in chemical and optical physics to treat rigorously the interaction of electromagnetic fields with matter. It is also widely used to evaluate intermolecular interactions. The multipolar version of the Hamiltonian is commonly obtained by carrying out a unitary transformation of the Coulomb gauge Hamiltonian that goes by the name of Power-Zienau-Woolley (PZW). Not only does the formulation provide excellent agreement with experiment, and versatility in its predictive ability, but also superior physical insight. Recently, the foundations and validity of the PZW Hamiltonian have been questioned, raising a concern over issues of gauge transformation and invariance, and whether observable quantities obtained from unitarily equivalent Hamiltonians are identical. Here, an in-depth analysis of theoretical foundations clarifies the issues and enables misconceptions to be identified. Claims of non-physicality are refuted: the PZW transformation and ensuing Hamiltonian are shown to rest on solid physical principles and secure theoretical ground

Evidence for a Semisolid Phase State of Aerosols and Droplets Relevant to the Airborne and Surface Survival of Pathogens

The phase state of respiratory aerosols and droplets has been linked to the humidity-dependent survival of pathogens such as SARS-CoV-2. To inform strategies to mitigate the spread of infectious disease, it is thus necessary to understand the humidity-dependent phase changes associated with the particles in which pathogens are suspended. Here, we study phase changes of levitated aerosols and droplets composed of model respiratory compounds (salt and protein) and growth media (organic-inorganic mixtures commonly used in studies of pathogen survival) with decreasing relative humidity (RH). Efflorescence was suppressed in many particle compositions and thus unlikely to fully account for the humidity-dependent survival of viruses. Rather, we identify organic-based, semisolid phase states that form under equilibrium conditions at intermediate RH (45 to 80%). A higher-protein content causes particles to exist in a semisolid state under a wider range of RH conditions. Diffusion and, thus, disinfection kinetics are expected to be inhibited in these semisolid states. These observations suggest that organic-based, semisolid states are an important consideration to account for the recovery of virus viability at low RH observed in previous studies. We propose a mechanism in which the semisolid phase shields pathogens from inactivation by hindering the diffusion of solutes. This suggests that the exogenous lifetime of pathogens will depend, in part, on the organic composition of the carrier respiratory particle and thus its origin in the respiratory tract. Furthermore, this work highlights the importance of accounting for spatial heterogeneities and time-dependent changes in the properties of aerosols and droplets undergoing evaporation in studies of pathogen viability

Cordycepin, a metabolite of Cordyceps militaris, reduces immune-related gene expression in insects

Hypocrealean entomopathogenic fungi (EPF) (Sordariomycetes, Ascomycota) are natural regulators of insect populations in terrestrial environments. Their obligately-killing life-cycle means that there is likely to be strong selection pressure for traits that allow them to evade the effects of the host immune system. In this study, we quantified the effects of cordycepin (3’-deoxyadenosine), a secondary metabolite produced by Cordyceps militaris (Hypocreales, Cordycipitaceae), on insect susceptibility to EPF infection and on insect immune gene expression. Application of the immune stimulant curdlan (20 µg ml-1, linear beta-1,3-glucan, a constituent of fungal cell walls) to Drosophila melanogaster S2r+ cells resulted in a significant increase in the expression of the immune effector gene metchnikowin compared to a DMSO-only control, but there was no significant increase when curdlan was co-applied with 25 µg ml-1 cordycepin dissolved in DMSO. Injection of cordycepin into larvae of Galleria mellonella (Lepidoptera: Pyralidae) resulted in dose-dependent mortality (LC50 of cordycepin = 2.1 mg per insect 6 days after treatment). Incubating conidia of C. militaris and Beauveria bassiana (Hypocreales, Cordycipitaceae; an EPF that does not synthesize cordycepin) with 3.0 mg ml-1 cordycepin had no effect on the numbers of conidia germinating in vitro. Co-injection of G. mellonella with a low concentration of cordycepin (3.0 mg ml-1 ) plus 10 or 100 conidia per insect of C. militaris or B. bassiana caused a significant decrease in insect median survival time compared to injection with the EPF on their own. Analysis of predicted vs. observed mortalities indicated a synergistic interaction between cordycepin and the EPF. The injection of C. militaris and B. bassiana into G. mellonella resulted in increased expression of the insect immune effector genes lysozyme, IMPI and gallerimycin at 72 h post injection, but this did not occur when the EPF were co-injected with 3.0 mg ml-1 cordycepin. In addition, we observed increased expression of IMPI and lysozyme at 48 h after injection with C. militaris, B. bassiana and sham injection (indicating a wounding response), but this was also prevented by application of cordycepin. These results suggest that cordycepin has potential to act as a suppressor of the immune response during fungal infection of insect hosts