Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity.

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-ß promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-ß transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the µM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.

[Continuous epidural anesthesia with a double catheter for sedation for surgery of the vertebral dorsolumbar column]. / Anestesia epidurale continua mediante doppio catetere più sedazione per la chirurgia della colonna vertebrale dorso-lombare.

The case of presented of an obese 42 year-old female patient, undergoing a second spinal cord decompression for a large dorsolumbar fibrous-scar mass, having a small-bore Montgomery tracheal stent (T-tube) on site. Stent replacement with a tracheotomy tube was impossible because of strong accretions hindering stent removal, as well as insertion of a suitable tracheal tube through the external stent branch, because of its very small lumen. So, general anaesthesia was not administered and surgery was performed under continuous epidural block, with light sedation, using two catheters introduced up and down the compression. Surgical and anaesthetic outcomes were optimal and confirm the effectiveness and safety of epidural anaesthesia for dorsolumbar spinal surgery, even in the obese patient. Moreover, with the continuous double-catheter technique it was possible to achieve a good and homogeneous spread of the analgesic solution, with low volume, despite the hard compression and deformation of the epidural space due to the fibrous-scar mass.

Phosphorylated and non-phosphorylated neurofilament epitopes are co-distributed in the fish Mauthner axon.

The neurofilament (NF) polypeptides of the fish giant Mauthner cell axons (MAs) and their degree of phosphorylation were investigated in the adult by means of immunoblot and immunohistochemical staining. Fasciculus longitudinalis medialis (FLM) axons of much smaller caliber, among which MAs run in the ventral spinal cord, were also analyzed in two teleost fish belonging to continuously growing species. To detect NF polypeptide subunits, commercially available monoclonal antibodies against mammalian NF-L (68 kDa), NF-M (160 kDa), phosphorylated (P) and non-phosphorylated (nP) NF-H (200 kDa) epitopes were used. These antibodies labelled bands of the identical molecular weight in fish cervical spinal cord total protein immunoblots. A peculiar NF composition of the MAs was observed, following immunohistochemical staining i.e. a low NF-M expression and a codistribution of P and (nP)-NF-H epitopes. Moreover, on the basis of immunoperoxidase staining in ultrathin longitudinal MA sections, we suggest that P NF-H are arranged in bundles, whilst (nP)-NF-H are likely to be free in the axoplasm. By contrast, FLM axons were found reactive with antibodies only against P NF-H. These results confirm that in carp and trout, NF have epitopes cross-reacting with monoclonal antibodies directed against the mammalian NF subunits. Furthermore, as regards the co-distribution of phosphorylated and non-phosphorylated NF-H epitopes in the M-cell axons, these might be considered as not yet completely mature axons taking into account that carp and trout belong to continuously growing species.

PCNA/cyclin expression and BrdU uptake define proliferating myosatellite cells during hyperplastic muscle growth of fish (Cyprinus carpio L.).

The possibility of detecting in situ proliferating myosatellite cells during postlarval muscle growth in the carp (Cyprinus carpio, L.) by means of BrdU and PCNA (Cyclin) immunohistochemistry has been evaluated on paraffin embedded sections. Nine subadult stages were defined according to the body length and weight. The fish were injected intraperitoneally with BrdU and fixed one hour later. Adjacent cross sections mounted on glass slides were incubated with monoclonal antiBrdU (1:100) and antiPCNA (1:200) antibody. The proliferative rate, defined as the percentage of labelled cells for each stage, was correlated to the corresponding percentage of small fibers (area less than 200 microns 2) determined by morphometric analysis. Desmin expression, immunocytochemically detected, was aimed at discriminating between the postmitotic and stem myosatellite cells. A low myosatellite cell proliferative activity (2-7%) throughout the carp growth period considered was demonstrated. Quantitative analysis provided evidence that during growth stages in which small fibers are numerous, the myosatellite cell proliferative activity is low and it increases when the small fibers number decreases. It is suggested that an age dependence of myosatellite cell proliferative rate controls the recruitment of new fibers in the carp.