Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His ₆ -tagged C-terminal peptide carrying several post-translational modifications at Cys ⁵³⁸ such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD( _319-541 )-(His) ₆ ] ₂ linked by an intermolecular disulfide bond (Cys ₅₃₈ -Cys ₅₃₈ ) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

Efficient resolution of neuroinflammation and debris clearance are key determinants of successful central nervous system (CNS) regeneration. Regenerative vertebrates such as Danio rerio often show faster immune resolution and debris clearance than mammals, yet the molecular determinants underlying these differences remain incompletely understood. TAM receptor tyrosine kinases (Tyro3, Axl, and Mertk) and their ligands Gas6 and Protein S are central regulators of phagocytosis and immune resolution in the nervous system, but whether intrinsic structural properties of these receptor–ligand complexes contribute to regenerative efficiency has not been systematically explored. Here, we perform a comparative in silico analysis of TAM receptors and ligands from zebrafish, human, and mouse, integrating sequence evolution, high-confidence structural modeling, interface characterization, and electrostatic analysis. Despite substantial sequence divergence between mammals and zebrafish, ligand-binding domains retain strong structural conservation, supporting a conserved global mode of TAM–ligand engagement. At the interface level, zebrafish complexes exhibit enhanced electrostatic contributions and increased salt-bridge density, particularly in the Tyro3–Protein S interaction. Residue-resolved electrostatic analysis identifies clustered interface hotspots that are conserved in spatial organization and physicochemical function across species, despite evolutionary rewiring of individual contacts. Together, these findings suggest that TAM receptor–ligand interfaces are evolutionarily tuned through subtle electrostatic and geometric optimization rather than large-scale structural changes. This conserved yet adaptable electrostatic framework supports a mechanistic hypothesis in which conserved TAM receptor architecture permits species-specific electrostatic tuning of receptor–ligand interfaces, potentially contributing to differences in TAM-dependent phagocytic signaling efficiency across vertebrates

Protein sequences are commonly interpreted through biochemical and evolutionary lenses, emphasizing structure-function relationships and selection in sequence space. Here we develop a complementary viewpoint: proteins as statistical languages, strings over a finite alphabet generated by constrained stochastic processes. We formalize intrinsic informational descriptors of protein ensembles, including composition entropy H1, adjacent mutual information I1, and separation-dependent information profiles Id. A null-model ladder (uniform, composition-matched i.i.d., and Markov-1) separates compositional effects from genuine positional dependence. We then evaluate these descriptors empirically across 20 UniProt reference proteomes spanning major clades, using protein-level bootstrap resampling and matched synthetic controls. Real proteomes consistently depart from composition-matched i.i.d. baselines and exhibit information profiles that remain elevated beyond the decay expected under first-order Markov surrogates, indicating dependencies beyond local transition statistics. Finally, a compressibility proxy (gzip) provides an orthogonal signature of redundancy relative to i.i.d. controls at matched composition. Together, these results support the view of proteomes as constrained statistical languages and provide model-agnostic fingerprints for comparing sequence ensembles. These signatures provide a lightweight diagnostic layer for comparing proteomes prior to mechanistic modeling.

Comparative studies increasingly score amino-acid variation across species with structure-based predictors applied to a single structural template, but whether the template itself can create associations with species traits has received little attention. We examined this while testing whether long-lived species carry proteins predicted to be more stable or, under nearly neutral theory, less stable. In two independent tetrapod radiations, 57 placental mammals (1,755 genes) and 40 reptiles and amphibians (1,910 genes), we reconstructed ancestral proteins, predicted with ThermoMPNN the folding effect of every amino-acid difference between the ancestor and each species on AlphaFold backbones, and related the mean effect to lifespan by phylogenetic regression, following analysis plans fixed in advance. With a human backbone, primates, which are long-lived for their size, appeared least destabilized, producing a weak apparent association with lifespan. In a pre-specified confirmatory test, a platypus backbone reduced the primate offset from − 1.96 to − 0.62 SD and the association disappeared ( P = 0.701). In reptiles, a human backbone, although equidistant in time, favoured slowly evolving turtles and crocodilians and gave a nominal association that two fish backbones did not reproduce. The predictor captured site-level stability signal, yet with outgroup templates neither radiation showed a robust association between predicted stability and longevity, although moderate effects cannot be excluded. The choice of structural template can therefore create apparent cross-species associations. We propose four checks to detect them: ancestral references, outgroup templates, tests for clade and rate effects, and tests of dependence on a few clades.

The gut microbiota–brain axis has emerged as a key regulator of central nervous system (CNS) development and function. Beyond its established roles in immunity and behavior, growing evidence indicates that microbial-derived metabolites influence glial biology, including oligodendrocyte development and myelination. In this review, we synthesize current findings supporting a role for microbial metabolites—particularly short-chain fatty acids (SCFAs), tryptophan-derived indoles, and secondary bile acids—in regulating oligodendrocyte precursor cell (OPC) proliferation, differentiation, and myelin formation. We examine the molecular and cellular mechanisms involved, including epigenetic regulation through histone deacetylase inhibition, G-protein–coupled receptor signaling, nuclear receptor activation, and metabolic support pathways. Experimental and clinical studies indicate that butyrate modulates oligodendrocyte lineage progression via chromatin remodeling, indole derivatives signal through the aryl hydrocarbon receptor (AhR) to attenuate neuroinflammation and promote glial homeostasis, and secondary bile acids act through receptors such as TGR5 and the vitamin D receptor to support pro-remyelinating environments. Additionally, microbial acetate contributes to acetyl-CoA pools necessary for lipid biosynthesis and epigenetic regulation within oligodendrocytes. Notably, dysbiosis and reduced availability of microbial metabolites have been consistently reported in Multiple Sclerosis (MS), a demyelinating disorder characterized by impaired remyelination, suggesting that microbiome-targeted strategies—including dietary interventions and postbiotic supplementation—may complement existing therapies. Collectively, current evidence supports the concept that microbial metabolites function as systemic modulators of CNS myelination, linking dietary and microbial inputs to oligodendrocyte biology and highlighting promising avenues for translational research in demyelinating diseases.