CBD attenuates the psychotic-like effects of cannabis over time in recreational users. Higher THC negatively impacts on memory and psychological well-being. These findings raise concerns for the harms stemming from use of varieties such as 'skunk' (sensimillia), which lack any CBD but currently dominate the supply of cannabis in many countries.

AimsIn the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca2+ oscillations that can cause lethal cell injury. These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca2+ oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels (TPCs, TPC1 and 2).Methods and resultsAn NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed Ca2+ oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury.ConclusionNAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury.

Surface patterning (micro-moulding) of dense, biomimetic collagen is a simple tool to produce complex tissues using layer-by-layer assembly. The aim here was to channelise three-dimensional constructs for improved perfusion. Firstly, collagen fibril accumulation was measured by comparative image analysis to understand the mechanisms of structure formation in plastically compressed collagen during µ-moulding. This showed that shape (circular or rectangular) and dimensions of the template affected collagen distribution around moulded grooves and consequently their stability. In the second part, this was used for effective fabrication of multi-layered plastically compressed collagen constructs with internal channels by roofing the grooves with a second layer. Using rectangular templates of 25/50/100 µm widths and 75 µm depth, grooves were µ-moulded into the fluid-leaving surface of collagen layers with predictable width/depth fidelities. These grooves were then roofed by addition of a second plastically compressed collagen layer on top to produce µ-channels. Resulting µ-channels retained their dimensions and were stable over time in culture with fibroblasts and could be cell seeded with a lining layer by simple transfer of epithelial cells. The results of this study provide a valuable platform for rapid fabrication of complex collagen-based tissues in particular for provision of perfusing microchannels through the bulk material for improved core nutrient supply.

Reperfusion of ischaemic cells causes intracellular Ca2+ oscillations as the sarcoplasmic reticulum (SR) takes up and releases Ca2+, leading to hypercontracture and cell death. In other systems, nicotinic acid adenine nucleotide phosphate (NAADP) acts as a second messenger to stimulate Ca2+ release from acidic intracellular Ca2+ stores, which in turn triggers Ca2+ release from the SR. We hypothesised that NAADP signalling is involved in the Ca2+ fluctuations that occur at reperfusion.We examined the effects of a novel NAADP inhibitor, Ned-19, on ischaemia-reperfusion injury in isolated adult rat ventricular cardiomyocytes (ARVC). The sensitivity of mitochondrial permeability transition pore (mPTP) was measured in ARVC using a laser-induced oxidative stress model. SR Ca2+ release was measured by treating cells loaded with fluorescent dye, fluo-4-AM, with caffeine. Cardioprotection was tested by exposing ARVC to metabolic ischaemia-reperfusion. Ned-19 was found to significantly delay the time to mPTP opening by 76%±16%, 55%±20%, 47%±19% and 44%±17% (all p<0.05) at concentrations of 100 μmol/l, 10 μmol/l, 1 μmol/l and 0.1 μmol/l, respectively, compared with the control group. Concentrations of Ned-19 at 100 μmol/l, 10 μmol/l and 1 μmol/l, but not 0.1 μmol/l, significantly inhibited caffeine-stimulated SR Ca2+ release (71.6%±2.0%, 34.2%±1.9%, 55.6%±5.5% and −14%±21%, respectively) indicating non-specific effects at higher concentrations. A low dose of 0.1 μmol/l Ned-19 increased the survival of cells following metabolic ischaemia-reperfusion to 46%±19% from 29% (control).In conclusion, we have shown the involvement of NAADP in SR Ca2+ release and mPTP opening, and that by inhibiting NAADP signalling at reperfusion with Ned-19, cardiomyocytes may be protected against ischaemia-reperfusion injury.

IntroductionNorovirus is the most common cause of viral gastroenteritis in man. It affects approximately 267 million people/annum and, although usually self-limiting, infection is still associated with around 200,000 deaths/annum. Norovirus infection has a significant, detrimental impact on societal infrastructure; it is the leading pathogen responsible for forced-ward closures in the NHS. There are no specific treatments available; the most promising target for antiviral therapy is noroviral 3 C protease (3 CLpro) which processes the polyprotein essential for the production of viral proteins. Inhibiting 3 CLpro would stop viral replication. This study focused on the function of 3 CLpro, especially the cleavage sites from its precursor.MethodsThe wild-type (WT) 3 CLpro sequence was altered with a cysteine to alanine base substitution to create a 3 CLpro mutant with greatly reduced catalytic activity. The mutant was expressed in E.coli and purified using ion exchange and size-filtration chromatography. Protein expression was confirmed by gel electrophoresis and Western blotting. The specificity of 3 CLpro was studied using a spectrophotometric assay and the rates of reaction of mutant and WT 3 CLpro with substrate were compared. The chemical composition of mutant and WT 3 CLpro were examined using mass spectroscopy.ResultsWestern blot analysis showed multiple bands indicating that both WT and mutant 3 CLpro appeared to be cleaved out of their precursor. Enzyme kinetic studies, however, confirmed that mutant 3 CLpro had negligible catalytic activity; the WT 3 CLpro’s turnover rate of catalytic activity was 25 times faster than that of mutant 3 CLpro. Mass spectrometry of mutant 3 CLpro generated a mass spectrum and transformed to a protein mass of 18,747.5. This confirmed the identity of noroviral 3 CLpro, which is 19 kDA.ConclusionMutant 3 CLpro was still cleaved out of its precursor despite the fact that it has negligible catalytic activity. The most likely explanation is that the cleavage was effected by an E.coli protease, possibly a metalloprotease, acting at the upstream and downstream boundaries thereby releasing the processed mutant 3 CLpro. The fact that mutant 3 CLpro can be cleaved out of its precursor by host-cell proteases raises the possibility that WT 3 CLpro could also be processed by exogenous proteases, rather than cleaving itself from its precursor. If this action by host cell proteases occurs in vivo, then it might indicate that the norovirus 3 CLpro is only needed for the cleavage of polyproteins within the newly formed virions, which do not have access to host cell proteases. This has significant implications for future research.Disclosure of InterestNone Declared