Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer’s disease.pre-print2,41 M

Levosimendan for resuscitating the microcirculation in patients with septic shock: A randomized controlled study

__Introduction:__ The purpose of the present study was to investigate microcirculatory blood flow in patients with septic shock treated with levosimendan as compared to an active comparator drug (i.e. dobutamine). The primary end point was a difference of ≥ 20% in the microvascular flow index of small vessels (MFIs) among groups. __Methods:__ The study was designed as a prospective, randomized, double-blind clinical trial and performed in a multidisciplinary intensive care unit. After achieving normovolemia and a mean arterial pressure of at least 65 mmHg, 40 septic shock patients were randomized to receive either levosimendan 0.2 μg·kg-1·min-1(n = 20) or an active comparator (dobutamine 5 μg·kg-1·min-1; control; n = 20) for 24 hours. Sublingual microcirculatory blood flow of small and medium vessels was assessed by sidestream dark-field imaging. Microcirculatory variables and data from right heart catheterization were obtained at baseline and 24 hours after randomization. Baseline and demographic data were compared by means of Mann-Whitney rank sum test or chi-square test, as appropriate. Microvascular and hemodynamic variables were analyzed using the Mann-Whitney rank sum test. __Results:__ Microcirculatory flow indices of small and medium vessels increased over time and were significantly higher in the levosimendan group as compared to the control group; P = .02; MFIs 2.9. The relative increase of perfused vessel density vs. baseline was significantly higher in the levosimendan group than in the control group. In addition, the heterogeneity index decreased only in the levosimendan group. There was no statistically significant correlation between systemic and microcirculatory flow variables within each group. __Conclusions:__ Compared to a standard dose of 5 μg·kg-1·min-1of dobutamine, levosimendan at 0.2 μg·kg-1·min-1improved sublingual microcirculatory blood flow in patients with septic shock, as reflected by changes in microcirculatory flow indices of small and medium vessels.Trial registration: NCT00800306

Cannabinoid CB2 Receptors Modulate Microglia Function and Amyloid Dynamics in a Mouse Model of Alzheimer's Disease

The distribution and roles of the cannabinoid CB2 receptor in the CNS are still a matter of debate. Recent data suggest that, in addition to its presence in microglial cells, the CB2 receptor may be also expressed at low levels, yet biologically relevant, in other cell types such as neurons. It is accepted that the expression of CB2 receptors in the CNS is low under physiological conditions and is significantly elevated in chronic neuroinflammatory states associated with neurodegenerative diseases such as Alzheimer's disease. By using a novel mouse model (CB2EGFP/f/f), we studied the distribution of cannabinoid CB2 receptors in the 5xFAD mouse model of Alzheimer's disease (by generating 5xFAD/CB2EGFP/f/f mice) and explored the roles of CB2 receptors in microglial function. We used a novel selective and brain penetrant CB2 receptor agonist (RO6866945) as well as mice lacking the CB2 receptor (5xFAD/CB2-/-) for these studies. We found that CB2 receptors are expressed in dystrophic neurite-associated microglia and that their modulation modifies the number and activity of microglial cells as well as the metabolism of the insoluble form of the amyloid peptide. These results support microglial CB2 receptors as potential targets for the development of amyloid-modulating therapies.Funding The present work has been supported by a grant from Ministerio de Ciencia e Innovacion (ref PID2019-108992RB-I00 and ref PID2019-107548RB-I00) to JR and PG, respectively, by the Basque Government (ref IT1230-19) to PG, and the Research and Education Component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin to CJH

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

The neutron and its role in cosmology and particle physics

Experiments with cold and ultracold neutrons have reached a level of precision such that problems far beyond the scale of the present Standard Model of particle physics become accessible to experimental investigation. Due to the close links between particle physics and cosmology, these studies also permit a deep look into the very first instances of our universe. First addressed in this article, both in theory and experiment, is the problem of baryogenesis ... The question how baryogenesis could have happened is open to experimental tests, and it turns out that this problem can be curbed by the very stringent limits on an electric dipole moment of the neutron, a quantity that also has deep implications for particle physics. Then we discuss the recent spectacular observation of neutron quantization in the earth's gravitational field and of resonance transitions between such gravitational energy states. These measurements, together with new evaluations of neutron scattering data, set new constraints on deviations from Newton's gravitational law at the picometer scale. Such deviations are predicted in modern theories with extra-dimensions that propose unification of the Planck scale with the scale of the Standard Model ... Another main topic is the weak-interaction parameters in various fields of physics and astrophysics that must all be derived from measured neutron decay data. Up to now, about 10 different neutron decay observables have been measured, much more than needed in the electroweak Standard Model. This allows various precise tests for new physics beyond the Standard Model, competing with or surpassing similar tests at high-energy. The review ends with a discussion of neutron and nuclear data required in the synthesis of the elements during the "first three minutes" and later on in stellar nucleosynthesis.Comment: 91 pages, 30 figures, accepted by Reviews of Modern Physic

Water-Compatible Cycloadditions of Oligonucleotide-Conjugated Strained Allenes for DNA-Encoded Library Synthesis

DNA-encoded libraries of small molecules are being explored extensively for the identification of binders in early drug-discovery efforts. Combinatorial syntheses of such libraries require water- and DNA-compatible reactions, and the paucity of these reactions currently limit the chemical features of resulting barcoded products. The present work introduces strain-promoted cycloadditions of cyclic allenes under mild conditions to DNA-encoded library synthesis. Owing to distinct cycloaddition modes of these reactive intermediates with activated olefins, 1,3-dipoles and dienes, the process generates diverse molecular architectures from a single precursor. The resulting DNA-barcoded compounds exhibit unprecedented ring and topographic features—related to elements found to be powerful in phenotypic screening

Highly Selective, Amine-Derived Cannabinoid Receptor 2 Probes

The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB2R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood—likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB2R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB2R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB1R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB2R to date

Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells

ISSN:0002-7863ISSN:1520-512