Allosteric modulation of G-protein–coupled receptors represents a key goal of current pharmacology. In particular, endogenous allosteric modulators might represent important targets of interventions aimed at maximizing therapeutic efficacy and reducing side effects of drugs. Here we show that the anti-inflammatory lipid lipoxin A 4 is an endogenous allosteric enhancer of the CB 1 cannabinoid receptor. Lipoxin A 4 was detected in brain tissues, did not compete for the orthosteric binding site of the CB 1 receptor (vs. 3 H-SR141716A), and did not alter endocannabinoid metabolism (as opposed to URB597 and MAFP), but it enhanced affinity of anandamide at the CB1 receptor, thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition, lipoxin A 4 displayed a CB 1 receptor-dependent protective effect against β-amyloid (1–40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB 1 receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTSDisrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1b release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1b, interferon-g and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONSColchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action -inhibition of pore formation induced by activation of P2X receptors -which could explain some of the anti-inflammatory effects of colchicine. LINKED ARTICLEThis article is commented on by Pelegrín, pp. 908-911 of this issue. To view this commentary visit http://dx.doi.org/10.1111/ j. 1476-5381.2011.01325.x Abbreviations DAPI, 4′,6-diamidino-2-phenylindole; DCFH2DA, dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; EB, ethidium bromide; IFN-g, interferon-g; IL-1b, interleukin-1b; LPS, lipopolysaccharide; PGP, P-glycoprotein; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species BJP British Journal of Pharmacology

transport mechanism of anionic dyes displayed by macrophages was also able to support dye efflux and, once activated at 37°C, it remained active at 4°C, whereas uptake of cationic dyes was temperature-dependent and unidirectional. Our results indicate that the mechanism of ATP e -induced dye uptake, usually called a 'permeabilization phenomenon' and associated with a 'permeabilization pore' can be ascribed to at least two distinct mechanisms in macrophages: a diffusional pathway, possibly associated with the 440 pS Z pores, and a cation uptake mechanism that is not diffusional and should be ascribed to an, as yet, unidentified transport mechanism.Key words: ATP, P2 receptor, P2X 7 , Permeabilization, Macrophage, Cation, Anion, Pore Journal of Cell Science 3262 extracellular concentration of the dye (Steinberg et al., 1987a), it has been proposed that the mechanism underlying the dye uptake phenomena is free diffusion through a permeabilization pore, and it has been assumed that cations and anions uses the same pathway. However, evidence from several different sources indicate the involvement of distinct mechanisms (North, 2002;Egan et al., 2006). Whole-cell patch-clamp recordings performed on cells transfected with P2X 7 receptors indicate a permeability shift from low to high M r molecules in the first seconds after stimulation by ATP e , while keeping the selectivity for cations (Virginio et al., 1999). In addition, although ATP e induces the uptake of both cationic and anionic dyes in macrophages (Steinberg et al., 1987a), the uptake of anions has not been reported either in HEK-293 cells or astrocytomas transfected with P2X 7 , neither has it been reported in lymphocytes (Wiley et al., 1993;Surprenant et al., 1996;Rassendren et al., 1997;Chessell et al., 1998;Ferrari et al., 2000;Paukert et al., 2002;Duan et al., 2003;Suadicani et al., 2006), suggesting that in these experimental situations, ATP e might not induce membrane permeabilization to anions.Based on cell-attached patch-clamping experiments demonstrating the opening of large cation-and anion-permeant channels (Z pores) induced by ATP e in macrophages, we have hypothesized that the permeabilization pore of macrophages could be distinct from the receptor itself, but coupled to P2X 7 through an unidentified signaling mechanism (Coutinho-Silva and Persechini, 1997;Persechini et al., 1998). This possibility has recently been corroborated by data showing that pannexin-1, a protein that forms large non-selective transmembrane channels (Barbe et al., 2006), is involved in the phenomenon of P2X 7 -associated, ATP e -induced membrane permeabilization (Pelegrin and Surprenant, 2006;Locovei et al., 2007). However, there have been no reports of any large and non-selective unitary channels similar to the Z pores in cells transfected with P2X 7 receptors under conditions of ATP einduced dye uptake. Moreover, pannexin-1 channels are expected to be permeable to both cations and anions (Bao et al., 2004;Locovei et al., 2006;Locovei et al., 2007), whereas there are no ...

The infection by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes major public health concern and economic burden. Although clinically approved drugs have been repurposed to treat individuals with 2019 Coronavirus disease (COVID-19), the lack of safety studies and limited efficiency as well jeopardize clinical benefits. Daclatasvir and sofosbuvir (SFV) are clinically approved direct-acting antivirals (DAA) against hepatitis C virus (HCV), with satisfactory safety profile. In the HCV replicative cycle, daclatasvir and SFV target the viral enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. These characteristics of the HCV and SARS-CoV-2 motivated us to further study the activity of daclatasvir and SFV against the new coronavirus. Daclatasvir consistently inhibited the production of infectious SARS-CoV-2 virus particles in Vero cells, in the hepatoma cell line HuH-7 and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 μM, respectively. Daclatasvir targeted early events during SARS-CoV-2 replication cycle and prevented the induction of IL-6 and TNF-α, inflammatory mediators associated with the cytokine storm typical of SARS-CoV-2 infection. Sofosbuvir, although inactive in Vero cells, displayed EC50 values of 6.2 and 9.5 μM in HuH-7 and Calu-3 cells, respectively. Our data point to additional antiviral candidates, in especial daclatasvir, among drugs overlooked for COVID-19, that could immediately enter clinical trials.

Background Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. Methods SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19. Results Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Conclusions Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

SARS-CoV-2 infection during pregnancy is not usually associated with significant adverse effects. However, in this study, we report a fetal death associated with mild COVID-19 in a 34-week-pregnant woman. The virus was detected in the placenta and in an unprecedented way in several fetal tissues. Placental abnormalities (MRI and anatomopathological study) were consistent with intense vascular malperfusion, probably the cause of fetal death. Lung histopathology also showed signs of inflammation, which could have been a contributory factor. Monitoring inflammatory response and coagulation in high-risk pregnant women with COVID-19 may prevent unfavorable outcomes, as shown in this case.

DOPA decarboxylase (DDC; aromatic-L-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, preincubated with L-DOPA for 1 h, increased 250-and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mM L-DOPA, washed and followed by incubation in the presence of 0.5 mM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with L-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 lM). The incubation of retinas in medium previously conditioned by retinapigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for L-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided L-DOPA is supplied to the tissue. It also shows that RPE is a potential source of L-DOPA early in development.

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.