Gunshot residue detection in stagnant water: SEM-EDX or ICP-MS? A preliminary study.

The identification of gunshot residue (GSR) on wounds enables the differentiation of entry and exit wounds. Unfortunately, studies analyzing GSR on degraded bodies have been poorly documented, and no data exist regarding GSR detection after stagnant water immersion. The aim of this preliminary experimental study was to detect GSR on wounds altered in stagnant water, using scanning electron microscopy coupled with energy-dispersive X (SEM-EDX) and inductively coupled plasma mass spectrometry (ICP-MS). Shots were performed on sheep limbs with a 22LR at a distance of 20 cm. The limbs were then submerged in stagnant water and analyzed on days 0, 6, and 14. SEM-EDX was performed on previously dehydrated wounds. For ICP-MS analysis, the wounds were rubbed with a cotton swab that was then analyzed. In the SEM studies, a higher number of particles were detected in entry wounds compared to exit wounds under every set of experimental conditions. Unfortunately, SEM-EDX failed to detect GSR particles, even on day 0. ICP-MS enabled the detection of Pb, Sb, and Ba at every stage with higher quantities on entry than in exit. These elements remained detectable following limb immersion. ICP-MS enabled differentiate entry from exit wounds, even after immersion in stagnant water. Nevertheless, when manually swabbing the wounds, quantities of matter collected is highly variable. ICP-MS is a more suitable technique than SEM-EDX for GSR identification of wounds after decomposition in stagnant water; however, standardization is needed.

G protein stoichiometry dictates biased agonism through distinct receptor-G protein partitioning.

Biased agonism at G protein coupled receptors emerges as an opportunity for development of drugs with enhanced benefit/risk balance making biased ligand identification a priority. However, ligand biased signature, classically inferred from ligand activity across multiple pathways, displays high variability in recombinant systems. Functional assays usually necessity receptor/effector overexpression that should be controlled among assays to allow comparison but this calibration currently fails. Herein, we demonstrate that Gα expression level dictates the biased profiling of agonists and, to a lesser extent of ß-blockers, in a Gα isoform- and receptor-specific way, depending on specific G protein activity in different membrane territories. These results have major therapeutic implications since they suggest that the ligand bias phenotype is not necessarily maintained in pathological cell background characterized by fluctuations in G protein expression. Thus, we recommend implementation of G protein stoichiometry as a new parameter in biased ligand screening programs.

Delineating biased ligand efficacy at 7TM receptors from an experimental perspective.

During the last 10 years, the concept of "biased agonism" also called "functional selectivity" swamped the pharmacology of 7 transmembrane receptors and paved the way for developing signaling pathway-selective drugs with increased efficacy and less adverse effects. Initially thought to select the activation of only a subset of the signaling pathways by the reference agonist, bias ligands revealed higher complexity as they have been shown to stabilize variable receptor conformations that associate with distinct signaling events from the reference. Today, one major challenge relies on the in vitro determination of the bias and classification of these ligands, as a prerequisite for future in vivo and clinical translation. In this review, current experimental considerations for the bias evaluation related to choice of the cellular model, of the signaling pathway as well as of the assays are presented and discussed.

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.