VAMP7j: A Splice Variant of Human VAMP7 That Modulates Neurite Outgrowth by Regulating L1CAM Transport to the Plasma Membrane.

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.

1-Piperidine Propionic Acid as an Allosteric Inhibitor of Protease Activated Receptor-2.

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.

Comparative Surface Electrostatics and Normal Mode Analysis of High and Low Pathogenic H7N7 Avian Influenza Viruses.

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.

Exogenous Players in Mitochondria-Related CNS Disorders: Viral Pathogens and Unbalanced Microbiota in the Gut-Brain Axis.

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.

Design and experimental validation of an optimized microalgae-bacteria consortium for the bioremediation of glyphosate in continuous photobioreactors.

Glyphosate will be banned from Europe by the end of 2022, but its widespread use in the last decades and its persistence in the environment require the development of novel remediation processes. In this work, a bacterial consortium was designed de novo with the aim to remove glyphosate from polluted water, supported by the oxygen produced by a microalgal species. To this goal, bioinformatics tools were employed to identify the bacterial strains from contaminated sources (Pseudomonas stutzeri; Comamonas odontotermitis; Sinomonas atrocyanea) able to express enzymes for glyphosate degradation, while the microalga Chlorella protothecoides was chosen for its known performances in wastewater treatment. To follow a bioaugmentation approach, the designed consortium was cultivated in continuous photobioreactors at increasing glyphosate concentrations, from 5 to 50 mg L-1, to boost its acclimation to the presence of the herbicide and its capacity to remove it from water. C. protothecoides tolerance to glyphosate was verified through batch experiments. Remarkably, steady state conditions were reached and the consortium was able to live as a community in the reactor. The consortium activity was validated in both synthetic and real wastewater, where glyphosate concentration was reduced by about 53% and 79%, respectively, without the detection of aminomethylphosphonic acid formation.

Nuclear and Cytoplasmatic Players in Mitochondria-Related CNS Disorders: Chromatin Modifications and Subcellular Trafficking.

Aberrant mitochondrial phenotypes are common to many central nervous system (CNS) disorders, including neurodegenerative and neurodevelopmental diseases. Mitochondrial function and homeostasis depend on proper control of several biological processes such as chromatin remodeling and transcriptional control, post-transcriptional events, vesicle and organelle subcellular trafficking, fusion, and morphogenesis. Mutation or impaired regulation of major players that orchestrate such processes can disrupt cellular and mitochondrial dynamics, contributing to neurological disorders. The first part of this review provides an overview of a functional relationship between chromatin players and mitochondria. Specifically, we relied on specific monogenic CNS disorders which share features with mitochondrial diseases. On the other hand, subcellular trafficking is coordinated directly or indirectly through evolutionarily conserved domains and proteins that regulate the dynamics of membrane compartments and organelles, including mitochondria. Among these "building blocks", longin domains and small GTPases are involved in autophagy and mitophagy, cell reshaping, and organelle fusion. Impairments in those processes significantly impact CNS as well and are discussed in the second part of the review. Hopefully, in filling the functional gap between the nucleus and cytoplasmic organelles new routes for therapy could be disclosed.

The Family Keeps on Growing: Four Novel Fungal OYEs Characterized.

Aiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter ß6-loop and a disordered capping subdomain.

3D Printed Graphene-PLA Scaffolds Promote Cell Alignment and Differentiation.

Traumas and chronic damages can hamper the regenerative power of nervous, muscle, and connective tissues. Tissue engineering approaches are promising therapeutic tools, aiming to develop reliable, reproducible, and economically affordable synthetic scaffolds which could provide sufficient biomimetic cues to promote the desired cell behaviour without triggering graft rejection and transplant failure. Here, we used 3D-printing to develop 3D-printed scaffolds based on either PLA or graphene@PLA with a defined pattern. Multiple regeneration strategies require a specific orientation of implanted and recruited cells to perform their function correctly. We tested our scaffolds with induced pluripotent stem cells (iPSC), neuronal-like cells, immortalised fibroblasts and myoblasts. Our results demonstrated that the specific "lines and ridges" 100 µm-scaffold topography is sufficient to promote myoblast and fibroblast cell alignment and orient neurites along with the scaffolds line pattern. Conversely, graphene is critical to promote cells differentiation, as seen by the iPSC commitment to neuroectoderm, and myoblast fusions into multinuclear myotubes achieved by the 100 µm scaffolds containing graphene. This work shows the development of a reliable and economical 3D-printed scaffold with the potential of being used in multiple tissue engineering applications and elucidates how scaffold micro-topography and graphene properties synergistically control cell differentiation.

A glimpse on metazoan ZNFX1 helicases, ancient players of antiviral innate immunity.

The human zinc finger NFX1-type containing 1 (ZNFX1) is an interferon-stimulated protein associated to the outer mitochondrial membrane, able to bind dsRNAs and interact with MAVS proteins, promoting type I IFN response in the early stage of viral infection. An N-terminal Armadillo (ARM)-type fold and a large helicase core (P-loop) and zinc fingers confer RNA-binding and ATPase activities to ZNFX1. We studied the phylogenetic distribution of metazoan ZNFX1s, ZNFX1 gene expression trends and genomic and protein signatures during viral infection of invertebrates. Based on 221 ZNFX1 sequences, we obtained a polyphyletic tree with a taxonomy-consistent branching at the phylum-level only. In metazoan genomes, ZNFX1 genes were found either in single copy, with up to some tens of exons in vertebrates, or in multiple copies, with one or a few exons and one of them sometimes encompassing most of the coding sequence, in invertebrates like sponges, sea urchins and mollusks. Structural analyses of selected ZNFX1 proteins showed high conservation of the helicase region (P-loop), an overall conserved region and domain architecture, an ARM-fold mostly traceable, and the presence of intrinsically disordered regions of varying length and position. The remarkable over-expression of ZNFX1 in bivalve and gastropod mollusks infected with dsDNA viruses underscores the antiviral role of ZNFX1, whereas nothing similar was found in virus-infected nematodes and corals. Whether the functional diversification reported in the C. elegans ZNFX1 occurs in other metazoan proteins remains to be established.

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model.

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2-Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

Bioremediation of Per- and Poly-Fluoroalkyl Substances (PFAS) by Synechocystis sp. PCC 6803: A Chassis for a Synthetic Biology Approach.

One of the main concerns in industrialized countries is represented by per- and poly-fluoroalkyl substances (PFAS), persistent contaminants hardly to be dealt with by conventional wastewater treatment processes. Phyco-remediation was proposed as a green alternative method to treat wastewater. Synechocystis sp. PCC6803 is a unicellular photosynthetic organism candidate for bioremediation approaches based on synthetic biology, as it is able to survive in a wide range of polluted waters. In this work, we assessed the possibility of applying Synechocystis in PFAS-enriched waters, which was never reported in the previous literature. Respirometry was applied to evaluate short-term toxicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), which did not affect growth up to 0.5 and 4 mg L-1, respectively. Continuous and batch systems were used to assess the long-term effects, and no toxicity was highlighted for both compounds at quite high concentration (1 mg L-1). A partial removal was observed for PFOS and PFOA, (88% and 37%, with removal rates of about 0.15 and 0.36 mg L-1 d-1, respectively). Measurements in fractionated biomass suggested a role for Synechocystis in the sequestration of PFAS: PFOS is mainly internalized in the cell, while PFOA is somehow transformed by still unknown pathways. A preliminary bioinformatic search gave hints on transporters and enzymes possibly involved in such sequestration/transformation processes, opening the route to metabolic engineering in the perspective application of this cyanobacterium as a new phyco-remediation tool, based on synthetic biology.

A conserved Neurite Outgrowth and Guidance motif with biomimetic potential in neuronal Cell Adhesion Molecules.

The discovery of conserved protein motifs can, in turn, unveil important regulatory signals, and when properly designed, synthetic peptides derived from such motifs can be used as biomimetics for biotechnological and therapeutic purposes. We report here that specific Ig-like repeats from the extracellular domains of neuronal Cell Adhesion Molecules share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which mediates homo- and heterophilic interactions crucial in neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can boost neuritogenesis, and this potential is also retained by peptides with recombinant sequences, when fitting the NOG sequence pattern. The NOG motif discovery not only provides one more tile to the complex puzzle of neuritogenesis, but also opens the route to new neural regeneration strategies via a tunable biomimetic toolbox.

Graphene-Based Scaffolds for Regenerative Medicine.

Leading-edge regenerative medicine can take advantage of improved knowledge of key roles played, both in stem cell fate determination and in cell growth/differentiation, by mechano-transduction and other physicochemical stimuli from the tissue environment. This prompted advanced nanomaterials research to provide tissue engineers with next-generation scaffolds consisting of smart nanocomposites and/or hydrogels with nanofillers, where balanced combinations of specific matrices and nanomaterials can mediate and finely tune such stimuli and cues. In this review, we focus on graphene-based nanomaterials as, in addition to modulating nanotopography, elastic modulus and viscoelastic features of the scaffold, they can also regulate its conductivity. This feature is crucial to the determination and differentiation of some cell lineages and is of special interest to neural regenerative medicine. Hereafter we depict relevant properties of such nanofillers, illustrate how problems related to their eventual cytotoxicity are solved via enhanced synthesis, purification and derivatization protocols, and finally provide examples of successful applications in regenerative medicine on a number of tissues.

Normal modes analysis and surface electrostatics of haemagglutinin proteins as fingerprints for high pathogenic type A influenza viruses.

BACKGROUND: Type A influenza viruses circulate and spread among wild birds and mostly consist of low pathogenic strains. However, fast genome variation timely results in the insurgence of high pathogenic strains, which when infecting poultry birds may cause a million deaths and strong commercial damage. More importantly, the host shift may concern these viruses and sustained human-to-human transmission may result in a dangerous pandemic outbreak. Therefore, fingerprints specific to either low or high pathogenic strains may represent a very important tool for global surveillance. RESULTS: We combined Normal Modes Analysis and surface electrostatic analysis of a mixed strain dataset of influenza A virus haemagglutinins from high and low pathogenic strains in order to infer specific fingerprints. Normal Modes Analysis sorted the strains in two different, homogeneous clusters; sorting was independent of clades and specific instead to high vs low pathogenicity. A deeper analysis of fluctuations and flexibility regions unveiled a special role for the 110-helix region. Specific sorting was confirmed by surface electrostatics analysis, which further allowed to focus on regions and mechanisms possibly crucial to the low-to-high transition. CONCLUSIONS: Evidence from previous work demonstrated that changes in surface electrostatics are associated with the evolution and spreading of avian influenza A virus clades, and seemingly involved also in the avian to mammalian host shift. This work shows that a combination of electrostatics and Normal Modes Analysis can also identify fingerprints specific to high and low pathogenicity. The possibility to predict which specific mutations may result in a shift to high pathogenicity may help in surveillance and vaccine development.

Protein electrostatics: From computational and structural analysis to discovery of functional fingerprints and biotechnological design.

Computationally driven engineering of proteins aims to allow them to withstand an extended range of conditions and to mediate modified or novel functions. Therefore, it is crucial to the biotechnological industry, to biomedicine and to afford new challenges in environmental sciences, such as biocatalysis for green chemistry and bioremediation. In order to achieve these goals, it is important to clarify molecular mechanisms underlying proteins stability and modulating their interactions. So far, much attention has been given to hydrophobic and polar packing interactions and stability of the protein core. In contrast, the role of electrostatics and, in particular, of surface interactions has received less attention. However, electrostatics plays a pivotal role along the whole life cycle of a protein, since early folding steps to maturation, and it is involved in the regulation of protein localization and interactions with other cellular or artificial molecules. Short- and long-range electrostatic interactions, together with other forces, provide essential guidance cues in molecular and macromolecular assembly. We report here on methods for computing protein electrostatics and for individual or comparative analysis able to sort proteins by electrostatic similarity. Then, we provide examples of electrostatic analysis and fingerprints in natural protein evolution and in biotechnological design, in fields as diverse as biocatalysis, antibody and nanobody engineering, drug design and delivery, molecular virology, nanotechnology and regenerative medicine.

Commitment of Autologous Human Multipotent Stem Cells on Biomimetic Poly-L-lactic Acid-Based Scaffolds Is Strongly Influenced by Structure and Concentration of Carbon Nanomaterial.

Nanocomposite scaffolds combining carbon nanomaterials (CNMs) with a biocompatible matrix are able to favor the neuronal differentiation and growth of a number of cell types, because they mimic neural-tissue nanotopography and/or conductivity. We performed comparative analysis of biomimetic scaffolds with poly-L-lactic acid (PLLA) matrix and three different p-methoxyphenyl functionalized carbon nanofillers, namely, carbon nanotubes (CNTs), carbon nanohorns (CNHs), and reduced graphene oxide (RGO), dispersed at varying concentrations. qRT-PCR analysis of the modulation of neuronal markers in human circulating multipotent cells cultured on nanocomposite scaffolds showed high variability in their expression patterns depending on the scaffolds' inhomogeneities. Local stimuli variation could result in a multi- to oligopotency shift and commitment towards multiple cell lineages, which was assessed by the qRT-PCR profiling of markers for neural, adipogenic, and myogenic cell lineages. Less conductive scaffolds, i.e., bare poly-L-lactic acid (PLLA)-, CNH-, and RGO-based nanocomposites, appeared to boost the expression of myogenic-lineage marker genes. Moreover, scaffolds are much more effective on early commitment than in subsequent differentiation. This work suggests that biomimetic PLLA carbon-nanomaterial (PLLA-CNM) scaffolds combined with multipotent autologous cells can represent a powerful tool in the regenerative medicine of multiple tissue types, opening the route to next analyses with specific and standardized scaffold features.

Pandemic Avian Influenza and Intra/Interhaemagglutinin Subtype Electrostatic Variation among Viruses Isolated from Avian, Mammalian, and Human Hosts.

Host jump can result in deadly pandemic events when avian influenza A viruses broaden their host specificity and become able to infect mammals, including humans. Haemagglutinin-the major capsid protein in influenza A viruses-is subjected to high rate mutations, of which several occur at its "head": the receptor-binding domain that mediates specific binding to host cell receptors. Such surface-changing mutations may lead to antigenically novel influenza A viruses hence in pandemics by host jump and in vaccine escape by antigenic drift. Changes in haemagglutinin surface electrostatics have been recently associated with antigenic drift and with clades evolution and spreading in H5N1 and H9N2 viruses. We performed a comparative analysis of haemagglutinin surface electrostatics to investigate clustering and eventual fingerprints among representative pandemic (H5 and H7) and nonpandemic (H4 and H6) avian influenza viral subtypes. We observed preferential sorting of viruses isolated from mammalian/human hosts among these electrostatic clusters of a subtype; however, sorting was not "100% specific" to the different clusters. Therefore, electrostatic fingerprints can help in understanding, but they cannot explain alone the host jumping mechanism.

Electrostatic Variation of Haemagglutinin as a Hallmark of the Evolution of Avian Influenza Viruses.

Avian influenza virus is a zoonotic agent that significantly impacts public health and the poultry industry. Monitoring viral evolution and spread is crucial for surveillance and tracing programmes, which are currently based on serological or DNA sequencing-phylogenetics analysis. However, virus-host interactions, antigenic drift and spreading of viral clades strongly depend on variation in the surface features of capsid proteins. We report here that in silico comparative structural analysis of haemagglutinin can reveal relevant evolutionary fingerprints, particularly when integrated with sequence-based analyses. Phylogenetic analyses of H9 viral strains from wild birds and poultry, performed with different methods, reliably led to clustering of viruses into five main groups. Subsequent comparison of structural features showed congruence between such clustering and surface electrostatic fingerprints. These latter fingerprints relate group-specific variations in electrostatic charges and isocontours to well-known haemagglutinin sites involved in the modulation of immune escape and host specificity. This work suggests that the integration of structural and sequence comparisons may enhance investigations of trends and relevant mechanisms in viral evolution.

Neuronal commitment of human circulating multipotent cells by carbon nanotube-polymer scaffolds and biomimetic peptides.

AIM: We aimed to set up a self-standing, biomimetic scaffold system able to induce and support per se neuronal differentiation of autologous multipotent cells. MATERIALS & METHODS: We isolated a population of human circulating multipotent cells (hCMCs), and used carbon nanotube/polymer nanocomposite scaffolds to mimic electrical/nanotopographical features of the neural environment, and biomimetic peptides reproducing axon guidance cues from neural proteins. RESULTS: hCMCs showed high degree of stemness and multidifferentiative potential; stimuli from the scaffolds and biomimetic peptides could induce and boost hCMC differentiation toward neuronal lineage despite the absence of exogenously added, specific growth factors. CONCLUSION: This work suggests the scaffold-peptides system combined with autologous hCMCs as a functional biomimetic, self-standing prototype for neural regenerative medicine applications.

Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway.

SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named 'phytolongins' (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the 'Phyl domain'. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway.