Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30 μM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants

Identification of Equid herpesvirus 2 in tissue-engineered equine tendon

Background: Incidental findings of virus-like particles were identified following electron microscopy of tissue-engineered tendon constructs (TETC) derived from equine tenocytes. We set out to determine the nature of these particles, as there are few studies which identify virus in tendons per se, and their presence could have implications for tissue-engineering using allogenic grafts. Methods: Virus particles were identified in electron microscopy of TETCs. Virion morphology was used to initially hypothesise the virus identity.  Next generation sequencing was implemented to identify the virus. A pan herpesvirus PCR was used to validate the RNASeq findings using an independent platform. Histological analysis and biochemical analysis was undertaken on the TETCs. Results: Morphological features suggested the virus to be either a retrovirus or herpesvirus. Subsequent next generation sequencing mapped reads to Equid herpesvirus 2 (EHV2). Histological examination and biochemical testing for collagen content revealed no significant differences between virally affected TETCs and non-affected TETCs. An independent set of equine superficial digital flexor tendon tissue (n=10) examined using designed primers for specific EHV2 contigs identified at sequencing were negative. These data suggest that EHV is resident in some equine tendon. Conclusions: EHV2 was demonstrated in equine tenocytes for the first time; likely from in vivo infection. The presence of EHV2 could have implications to both tissue-engineering and tendinopathy

Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation

Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance: Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract