Tracking the evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through genomic surveillance programs is undoubtedly one of the key priorities in the current pandemic situation. Although the genome of SARS-CoV-2 acquires mutations at a slower rate compared with other RNA viruses, evolutionary pressures derived from the widespread circulation of SARS-CoV-2 in the human population have progressively favored the global emergence, though natural selection, of several variants of concern that carry multiple non-synonymous mutations in the spike glycoprotein. These are often placed in key sites within major antibody epitopes and may therefore confer resistance to neutralizing antibodies, leading to partial immune escape, or otherwise compensate infectivity deficits associated with other non-synonymous substitutions. As previously shown by other authors, several emerging variants carry recurrent deletion regions (RDRs) that display a partial overlap with antibody epitopes located in the spike N-terminal domain (NTD). Comparatively, very little attention had been directed towards spike insertion mutations prior to the emergence of the B.1.1.529 (omicron) lineage. This manuscript describes a single recurrent insertion region (RIR1) in the N-terminal domain of SARS-CoV-2 spike protein, characterized by at least 49 independent acquisitions of 1-8 additional codons between Val213 and Leu216 in different viral lineages. Even though RIR1 is unlikely to confer antibody escape, its association with two distinct formerly widespread lineages (A.2.5 and B.1.214.2), with the quickly spreading omicron and with other VOCs and VOIs warrants further investigation concerning its effects on spike structure and viral infectivity.

Background Strongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to unusual auto-infective cycle of Strongyloides stercoralis. The lack of a diagnostic gold standard and limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers, with the result that our knowledge of S. stercoralis proteome remains limited. This study aims at expanding the characterization of S. stercoralis infective larvae (iL3) in order to further explore the mechanisms of parasitism and to highlight possible novel targets for serodiagnosis. Methods iL3 obtained from an infected subject were analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterization of the iL3 proteome we analysed the experimental dataset using an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction. Results Our pipeline identified 430 S. stercoralis proteins, 187 (43%) of which were uncharacterized. Oxidoreductases and peptidases were amongst the most represented protein categories, as highlighted by molecular function GO analyses, while membrane and mitochondrial proteins were the most represented cellular component GO categories. A high proportion of proteins bearing the CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families were also identified. Additionally, we highlighted nine proteins displaying low homology with H. sapiens or other related pathogens and bearing amino acid sequences with immunogenic properties. Conclusions Our comprehensive description and annotation of the S. stercoralis iL3 proteome contribute to expanding the ‘omics characterization of this parasite and provide experimental evidence on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Moreover, novel candidate immunogenic proteins to be evaluated as novel serological diagnostic markers are highlighted. Graphical Abstract

The translocator protein (TSPO) is a transmembrane protein present across the three domains of life. Its functional quaternary structure consists of one or more subunits. In mice, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

The translocator protein (TSPO) is a transmembrane protein present in the three domains of life. Its functional quaternary structure consists of one or more subunits. In mouse, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

The recent outbreak of the COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has shown the necessity for fast and broad drug discovery methods to enable us to react quickly to novel and highly infectious diseases. A well-known SARS-CoV-2 target is the viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication, which is essential for the viral life cycle. Here, we applied an interaction-based drug repositioning algorithm on all protein-compound complexes available in the protein database (PDB) to identify Mpro inhibitors and potential novel compound scaffolds against SARS-CoV-2. The screen revealed a heterogeneous set of 692 potential Mpro inhibitors containing known ones such as Dasatinib, Amodiaquine, and Flavin mononucleotide, as well as so far untested chemical scaffolds. In a follow-up evaluation, we used publicly available data published almost two years after the screen to validate our results. In total, we are able to validate 17% of the top 100 predictions with publicly available data and can furthermore show that predicted compounds do cover scaffolds that are yet not associated with Mpro. Finally, we detected a potentially important binding pattern consisting of 3 hydrogen bonds with hydrogen donors of an oxyanion hole within the active side of Mpro. Overall, these results give hope that we will be better prepared for future pandemics and that drug development will become more efficient in the upcoming years.