A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Effect of platform switching on peri-implant bone: A 3D finite element analysis.

STATEMENT OF PROBLEM: A consensus regarding the effects of platform switching on peri-implant marginal bone levels is lacking. Finite element studies have reported contradictory results. PURPOSE: The purpose of this finite element analysis study was to evaluate stress distribution in platform-switched (PS) and platform-matched (PM) implants and their surrounding bone. MATERIAL AND METHODS: An implant (4.5×11 mm) was modeled and screwed into a human mandibular bone block using a computer-aided design (CAD) software program. Two separate models were generated: (1) PM, 4.5-mm implant with 4.5-mm-wide abutment and (2) PS, 4.5-mm implant with 3.5-mm-wide abutment. Implant components were modeled with linear isotropic properties and bones with anisotropic properties. Vertical (200 to 800 N) and oblique (50 to 150 N) forces were applied to each model to simulate occlusal loads. Linear elastic analysis was performed using ANSYS Workbench 16. von Mises equivalent stresses in the implant assemblies and peri-implant bone were calculated and compared with independent samples t test (α=.05). RESULTS: von Mises equivalent stress values under simulated axial and nonaxial occlusal loads were lower for PM than for PS implant assemblies. However, the differences were not statistically significant. Stress within the peri-implant bone was significantly higher for the PM group than for the PS group (P<.001). CONCLUSIONS: Platform switching decreased stress within peri-implant bone and may help limit marginal bone resorption.

Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.

Ceramic Fracture in Metal-Ceramic Restorations: The Aetiology.

All dental restorations are liable to failure during function. Failure could be biologic, aesthetic, mechanical or a combination. Ceramic restorations in particular, including metal-ceramics, are prone to mechanical fracture, especially the fracture of veneering porcelain. Fracture of a metal-ceramic restoration jeopardizes function as well as aesthetics. It is equally onerous to manage for both patient and dentist. Optimal management of such cases requires a detailed knowledge of the aetiology behind this phenomenon. The current paper aims to highlight possible causative factors involved in the mechanical failures of metal-ceramic restorations. Clinical relevance: Ceramic fracture in metal-ceramic crowns and fixed partial dentures is routinely encountered in dental clinics. Knowledge of the aetiology is required to diagnose and manage such cases accurately as well as to avoid these errors in future.

Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A2A receptor (A2AAR), and connexin 43 (Cx43). Quantification of SV2A and A2AAR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

Interaction of Approved Drugs with Synaptic Vesicle Protein 2A.

Levetiracetam (LEV) and its recently approved derivative brivaracetam are anti-epileptic drugs with a unique mechanism of action. The synaptic vesicle protein 2A (SV2A) was previously identified as their main target. In the current study, we tested a collection of 500 approved drugs for interaction with the human SV2A protein expressed in Chinese hamster ovary cells. Competition binding studies were performed using cell lysates with high SV2A expression and [3 H]brivaracetam as a radioligand. A hit rate of 3% was obtained, defined as compounds that inhibited radioligand binding by more than 90% at a screening concentration of 20 µM. Subsequent concentration-inhibition curves revealed the antihistaminic prodrug loratadine (Ki = 1.16 µM) and the antimalarial drug quinine (Ki = 2.03 µM) to be the most potent SV2A protein ligands of the investigated drug library. Both compounds were similarly potent as LEV (Ki = 1.74 µM), providing structurally novel scaffolds for SV2A ligands. A pharmacophore model was established, which indicated steric and electronic conformities of brivaracetam with the new SV2A ligands, and preliminary structure-activity relationships were determined. The anti-convulsive effects of the natural product quinine may - at least in part - be explained by interaction with SV2A. Loratadine and quinine represent new lead structures for anti-epileptic drug development.