Francesco Filippini

Background: Vaccines development in this millennium started by the milestone work on Neisseria meningitidis B, reporting the invention of Reverse Vaccinology (RV), which allows to identify vaccine candidates (VCs) by screening bacterial pathogens genome or proteome through computational analyses. When NERVE (New Enhanced RV Environment), the first RV software integrating tools to perform the selection of VCs, was released, it prompted further development in the field. However, the problem-solving potential of most, if not all, RV programs is still largely unexploited by experimental vaccinologists that impaired by somehow difficult interfaces, requiring bioinformatic skills. Results: We report here on the development and release of NERVE 2.0 (available at: https://nerve-bio.org) which keeps the original integrative and modular approach of NERVE, while showing higher predictive performance than its previous version and other web-RV programs (Vaxign and Vaxijen). We renewed some of its modules and added innovative ones, such as Loop-Razor, to recover fragments of promising vaccine candidates or Epitope Prediction for the epitope prediction binding affinities and population coverage. Along with two newly built AI (Artificial Intelligence)-based models: ESPAAN and Virulent. To improve user-friendliness, NERVE was shifted to a tutored, web-based interface, with a noSQL-database to consent the user to submit, obtain and retrieve analysis results at any moment. Conclusions: With its redesigned and updated environment, NERVE 2.0 allows customisable and refinable bacterial protein vaccine analyses to all different kinds of users.

Background: Vaccines development in this millennium started by the milestone work on Neisseria meningitidis B , reporting the invention of Reverse Vaccinology (RV), which allows to identify vaccine candidates (VCs) by screening bacterial pathogens genome or proteome through computational analyses. When NERVE (New Enhanced RV Environment), the first RV software integrating tools to perform the selection of VCs, was released, it prompted further development in the field. However, the problem-solving potential of most, if not all, RV programs is still largely unexploited by experimental vaccinologists that impaired by somehow difficult interfaces, requiring bioinformatic skills. Results: We report here on the development and release of NERVE 2.0 (available at: https://nerve-bio.org ) which keeps the original integrative and modular approach of NERVE, while showing higher predictive performance than its previous version and other web-RV programs (Vaxign and Vaxijen). We renewed some of its modules and added innovative ones, such as Loop Razor , to recover fragments of promising vaccine candidates or epitopepredict for the epitope prediction binding affinities and population coverage. Along with two newly built AI (Artificial Intelligence)-based models: ESPAAN and Virulent. To improve user-friendliness, NERVE was shifted to a tutored, web-based interface, with a noSQL-database to consent the user to submit, obtain and retrieve analysis results at any moment. Conclusions: With its redesigned and updated environment, NERVE 2.0 allows customisable and refinable bacterial protein vaccine analyses to all different kinds of users.

Computer-aided analysis of proteins or nucleic acids seems like a matter of course nowadays; however, the history of Bioinformatics and Computational Biology is quite recent. The advent of high-throughput sequencing has led to the production of “big data”, which has also affected the field of virology. The collaboration between the communities of bioinformaticians and virologists already started a few decades ago and it was strongly enhanced by the recent SARS-CoV-2 pandemics. In this article, which is the first in a series on how bioinformatics can enhance virus research, we show that highly useful information is retrievable from selected general and dedicated databases. Indeed, an enormous amount of information—both in terms of nucleotide/protein sequences and their annotation—is deposited in the general databases of international organisations participating in the International Nucleotide Sequence Database Collaboration (INSDC). However, more and more virus-specific databases have been established and are progressively enriched with the contents and features reported in this article. Since viruses are intracellular obligate parasites, a special focus is given to host-pathogen protein-protein interaction databases. Finally, we illustrate several phylogenetic and phylodynamic tools, combining information on algorithms and features with practical information on how to use them and case studies that validate their usefulness. Databases and tools for functional inference will be covered in the next article of this series: Bioinformatics goes viral: II. Sequence-based and structure-based functional analyses for boosting virus research.

Antimicrobial resistance represents a major threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assays in vitro , we elucidated the molecular events governing the SOS response activation in P. aeruginosa , focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that let RecA trigger the autocleavage and inactivation of the LexA repressor. These results provide the groundwork for designing novel antimicrobial strategies and for exploring the potential translation of Escherichia coli -derived approaches, to address the health-threatening implications of bacterial infections. Graphical

The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity.

In the last decades, studies on the inflammatory signaling pathways in multiple pathological contexts have revealed new targets for novel therapies. Among the family of G-protein-coupled Proteases Activated Receptors, PAR2 was identified as a driver of the inflammatory cascade in many pathologies, ranging from autoimmune disease to cancer metastasis. For this reason, many efforts have been focused on the development of potential antagonists of PAR2 activity. This work focuses on a small molecule, 1-Piperidine Propionic Acid (1-PPA), previously described to be active against inflammatory processes, but whose target is still unknown. Stabilization effects observed by cellular thermal shift assay coupled to in-silico investigations, including molecular docking and molecular dynamics simulations, suggested that 1-PPA binds PAR2 in an allosteric pocket of the receptor inactive conformation. Functional studies revealed the antagonist effects on MAPKs signaling and on platelet aggregation, processes mediated by PAR family members, including PAR2. Since the allosteric pocket binding 1-PPA is highly conserved in all the members of the PAR family, the evidence reported here suggests that 1-PPA could represent a promising new small molecule targeting PARs with antagonistic activity.

In the last decades, studies on the inflammatory signaling pathways in multiple pathological contexts revealed new targets for novel therapies. Among the family of G-protein-coupled Proteases Activated Receptors, PAR2 was identified as a driver of the inflammatory cascade in many pathologies, ranging from autoimmune disease to cancer metastasis. For this reason, many efforts have been focused on the development of potential antagonists of PAR2 activity. This work focuses on a small molecule, 1-Piperidine Propionic Acid (1-PPA), previously described to be active against inflammatory processes, but whose target is still unknown. Stabilization effects observed by cellular thermal shift assay coupled to in-silico investigations, including molecular docking and Molecular Dynamics simulations, suggested that 1-PPA binds PAR2 in an allosteric pocket of the receptor inactive conformation. In addition, this compound was found able to inhibit platelet aggregation, a process mediated by PAR family members, including PAR2. Since the allosteric pocket binding 1-PPA is highly conserved in all the members of the PAR family, the evidence reported here suggests that 1-PPA could represent a promising new small molecule targeting PARs with antagonistic activity.

Influenza A viruses are rarely symptomatic in wild birds, while representing a higher threat to poultry and mammals, where they can cause a variety of symptoms, including death. H5 and H7 subtypes of influenza viruses are of particular interest because of their pathogenic potential and reported capacity to spread from poultry to mammals, including humans. The identification of molecular fingerprints for pathogenicity can help surveillance and early warning systems, which are crucial to prevention and protection from such potentially pandemic agents. In the past decade, comparative analysis of the surface features of hemagglutinin, the main protein antigen in influenza viruses, identified electrostatic fingerprints in the evolution and spreading of H5 and H9 subtypes. Electrostatic variation among viruses from avian or mammalian hosts was also associated with host jump. Recent findings of fingerprints associated with low and highly pathogenic H5N1 viruses, obtained by means of comparative electrostatics and normal modes analysis, prompted us to check whether such fingerprints can also be found in the H7 subtype. Indeed, evidence presented in this work showed that also in H7N7, hemagglutinin proteins from low and highly pathogenic strains present differences in surface electrostatics, while no meaningful variation was found in normal modes.

Billions of years of co-evolution has made mitochondria central to the eukaryotic cell and organism life playing the role of cellular power plants, as indeed they are involved in most, if not all, important regulatory pathways. Neurological disorders depending on impaired mitochondrial function or homeostasis can be caused by the misregulation of “endogenous players”, such as nuclear or cytoplasmic regulators, which have been treated elsewhere. In this review, we focus on how exogenous agents, i.e., viral pathogens, or unbalanced microbiota in the gut-brain axis can also endanger mitochondrial dynamics in the central nervous system (CNS). Neurotropic viruses such as Herpes, Rabies, West-Nile, and Polioviruses seem to hijack neuronal transport networks, commandeering the proteins that mitochondria typically use to move along neurites. However, several neurological complications are also associated to infections by pandemic viruses, such as Influenza A virus and SARS-CoV-2 coronavirus, representing a relevant risk associated to seasonal flu, coronavirus disease-19 (COVID-19) and “Long-COVID”. Emerging evidence is depicting the gut microbiota as a source of signals, transmitted via sensory neurons innervating the gut, able to influence brain structure and function, including cognitive functions. Therefore, the direct connection between intestinal microbiota and mitochondrial functions might concur with the onset, progression, and severity of CNS diseases.