Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the ßγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 µM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Experiment investigation on pulsed gamma-ray fluence rate effect on Yb-doped yttrium aluminum garnet scintillator.

As an ultrafast inorganic scintillator, Yb-doped Y3Al5O12 [yttrium aluminum garnet (YAG)] crystals have potential applications in various fields, such as ultrafast radiation detection, solar neutrino detection, pulsed radiation imaging, and nuclear reaction kinetics diagnosis. In this work, the fluence rate effect of pulsed γ rays on the Yb:YAG scintillation crystal was investigated at the "QiangGuang-I" facility. The experiment results show that the fluence rate linear response upper limit of the Yb:YAG crystal is about 9.1 × 1018 MeV cm-2 s-1. The Yb:YAG crystal changed from colorless to yellow, and the relative light output decreased to 63% of its initial value after the irradiations, which were attributed to the radiation induced damage. It is deduced that oxygen vacancies and divalent Yb cations were generated after the irradiations.

Ultrahigh brilliance quasi-monochromatic MeV γ-rays based on self-synchronized all-optical Compton scattering.

Inverse Compton scattering between ultra-relativistic electrons and an intense laser field has been proposed as a major route to generate compact high-brightness and high-energy γ-rays. Attributed to the inherent synchronization mechanism, an all-optical Compton scattering γ-ray source, using one laser to both accelerate electrons and scatter via the reflection of a plasma mirror, has been demonstrated in proof-of-principle experiments to produce a x-ray source near 100 keV. Here, by designing a cascaded laser wakefield accelerator to generate high-quality monoenergetic e-beams, which are bound to head-on collide with the intense driving laser pulse via the reflection of a 20-um-thick Ti foil, we produce tunable quasi-monochromatic MeV γ-rays (33% full-width at half-maximum) with a peak brilliance of ~3 × 10(22) photons s(-1) mm(-2) mrad(-2) 0.1% BW at 1 MeV. To the best of our knowledge, it is one order of magnitude higher than ever reported value of its kinds in MeV regime. This compact ultrahigh brilliance γ-ray source may provide applications in nuclear resonance fluorescence, x-ray radiology and ultrafast pump-probe nondestructive inspection.