SARS-CoV-2 variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication the original master mutant, named MB61°, was replaced by a minor quasispecies (MB61222) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB61°. Interestingly, co-infection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61222 over MB61° replication. This finding may be explained by a faster replicative activity of MB61222 as compared to MB61° as well as by the capability of MB61222 to induce peculiar viral RNA sensing mechanisms leading to an increased production of Interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB61° cannot escape this host antiviral response during MB61222 co-infection, since it has endocytosis as main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

Background. During the outbreak of coronavirus disease 2019 (COVID-19), allocating intensive care beds to patients needing acute care surgery became a very difficult task. Moreover, since general anesthesia is an aerosol-generating procedure, its use became controversial. This strongly restricted therapeutic strategies. Here, we report a series of undeferrable surgical cases treated with awake surgery under neuraxial anesthesia. Contextual benefits of this approach are deepened. Methods. During the first pandemic surge, thirteen patients (5 men and 8 women) with a mean age of 80 years, needing undelayable surgery due to abdominal emergencies, underwent awake open surgery at our Hospital. Prior to surgery, all patients underwent nasopharyngeal swab tests for COVID-19 diagnosis. In all cases, regional anesthesia (spinal, epidural, or combined spinal-epidural anesthesia) was performed. Intraoperative and postoperative pain intensities have been monitored and regularly assessed. A distinct pathway has been set up to keep patients of uncertain COVID-19 diagnosis separated from all other patients. Postoperative course has been examined. Results. The mean operative time was 87 minutes (minimum 60 minutes; maximum 165 minutes). In one case, conversion to general anesthesia was necessary. Postoperative pain was always well controlled. None of them required postoperative intensive care support. No perioperative major complications (Clavien–Dindo ≥3) occurred. Early readmission after surgery never occurred. All nasopharyngeal swabs resulted negative. Conclusions. In our experience, awake laparotomy under regional anesthesia resulted feasible, safe, painless, and, in specific cases, was the only viable option. This approach allowed prevention of the need of postoperative intensive monitoring during the COVID-19 era. In such a peculiar time, we believe it could become part of an ICU-preserving strategy and could limit viral transmission inside theatres.

Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing out the “bitter” and “sweet” aspects of this field of research. Furthermore, we attempt to bridge the gap between bioinformatics and glycobiology, which have so far been kept apart by conceptual and technical differences. For this purpose, we provide computational scientists and glycobiologists with the fundamentals of these two fields of research, with the aim of creating opportunities for their combined exploitation, and thereby contributing to a substantial improvement in scientific knowledge.

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants’ evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.

Mutations in the Myelin Protein Zero gene (MPZ), encoding P0, the major structural glycoprotein of peripheral nerve myelin, are the cause of Charcot–Marie-Tooth (CMT) type 1B neuropathy, and most P0 mutations appear to act through gain-of-function mechanisms. Here we investigated how misglycosylation, a pathomechanism encompassing several genetic disorders, may affect P0 function. Using in vitro assays, we showed that gain of glycosylation is more damaging for P0 trafficking and functionality as compared to loss of glycosylation. Hence, we generated, via CRISPR/Cas9, a mouse model carrying the MPZD61N mutation, predicted to generate a new N-glycosylation site in P0. In humans, MPZD61N causes a severe early onset form of CMT1B, suggesting that hyperglycosylation may interfere with myelin formation, leading to pathology. We show here that MPZD61N/+ mice develop a tremor as early as P15 which worsens with age and correlates with a significant motor impairment, reduced muscular strength and with substantial alterations in neurophysiology. Pathological analysis confirmed a dysmyelinating phenotype characterized by diffuse hypomyelination and focal hypermyelination. We find that the mutant P0D61N does not cause significant endoplasmic reticulum stress, a common pathomechanism in CMT1B, but is properly trafficked to myelin where it causes myelin uncompaction. Finally, we show that myelinating DRG cultures from MPZD61N mice replicate some of the abnormalities seen in vivo, suggesting that they may represent a valuable tool to investigate therapeutic approaches. Collectively our data indicate that the MPZD61N/+ mouse represents an authentic model of severe CMT1B affirming gain-of-glycosylation in P0 as a novel pathomechanism of disease.