A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.     

Electro-deposition on carbon black and carbon nanotubes of Pt nanostructured catalysts for methanol oxidation

An electrochemical method for the Pt nanoparticles deposition on porous and high surface carbon substrates (carbon black and carbon nanotubes), as an alternative way to prepare gas diffusion electrodes for polymer electrolyte fuel cells (PEFCs), is herein described. Pt nanoparticles well distributed and localized on the electrode surface were obtained by using an electric field. The electro-catalysts were prepared by single and multiple pulse galvanostatic polarizations in 1 M sulphuric acid + 5 mM exachloroplatinic acid solution. Chemical analysis, cyclic voltammetry and field emission gun scanning electron microscopy were used to determine the electrochemical features of Pt deposits and the influence of electro-deposition method on their nano-morphology. Electro-catalytic performances were studied by investigating the methanol oxidation reaction and the results are presented in form of surface specific activity and mass specific activity to take into account the electrochemical real surface and Pt loading. A comparison with commercial E-TEK Pt/C catalysts, prepared by traditional chemical reduction and heat treatment in hydrogen, shows that the electrodeposited catalyst presents higher activity at lower Pt loading.     

A Set of Dysregulated Target Genes to Reduce Neuroinflammation at Molecular Level

Increasing evidence links chronic neurodegenerative diseases with neuroinflammation; it is known that neuroprotective agents are capable of modulating the inflammatory processes, that occur with the onset of neurodegeneration pathologies. Here, with the intention of providing a means for active compounds’ screening, a dysregulation of neuronal inflammatory marker genes was induced and subjected to neuroprotective active principles, with the aim of selecting a set of inflammatory marker genes linked to neurodegenerative diseases. Considering the important role of microglia in neurodegeneration, a murine co-culture of hippocampal cells and inflamed microglia cells was set up. The evaluation of differentially expressed genes and subsequent in silico analysis showed the main dysregulated genes in both cells and the principal inflammatory processes involved in the model. Among the identified genes, a well-defined set was chosen, selecting those in which a role in human neurodegenerative progression in vivo was already defined in literature, matched with the rate of prediction derived from the Principal Component Analysis (PCA) of in vitro treatment-affected genes variation. The obtained panel of dysregulated target genes, including Cxcl9 (Chemokine (C-X-C motif) ligand 9), C4b (Complement Component 4B), Stc1 (Stanniocalcin 1), Abcb1a (ATP Binding Cassette Subfamily B Member 1), Hp (Haptoglobin) and Adm (Adrenomedullin), can be considered an in vitro tool to select old and new active compounds directed to neuroinflammation.     

Patients Recovering from Severe COVID-19 Develop a Polyfunctional Antigen-Specific CD4+ T Cell Response

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.     

Oxidation Stress as a Mechanism of Aging in Human Erythrocytes: Protective Effect of Quercetin

Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO₄²⁻ uptake, thiobarbituric acid reactive substances (TBARS) levels—a marker of lipid peroxidation—total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO₄²⁻ uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.     

Fabrication and properties of keratoses/polyvinyl alcohol blend films

Polymer Bulletin - Blend films of polyvinyl alcohol (PVA) and keratoses (KOx) were prepared using water as a common solvent. Aqueous solution of KOx from wool was extracted via oxidation using...     

Enhancing the Amyloid-β Anti-Aggregation Properties of Curcumin via Arene-Ruthenium(II) Derivatization

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder associated with severe dementia, progressive cognitive decline, and irreversible memory loss. Although its etiopathogenesis is still unclear, the aggregation of amyloid-β (Aβ) peptides into supramolecular structures and their accumulation in the central nervous system play a critical role in the onset and progression of the disease. On such a premise, the inhibition of the early stages of Aβ aggregation is a potential prevention strategy for the treatment of AD. Since several natural occurring compounds, as well as metal-based molecules, showed promising inhibitory activities toward Aβ aggregation, we herein characterized the interaction of an organoruthenium derivative of curcumin with Aβ(1–40) and Aβ(1–42) peptides, and we evaluated its ability to inhibit the oligomerization/fibrillogenesis processes by combining in silico and in vitro methods. In general, besides being less toxic to neuronal cells, the derivative preserved the amyloid binding ability of the parent compound in terms of equilibrium dissociation constants but (most notably) was more effective both in retarding the formation and limiting the size of amyloid aggregates by virtue of a higher hindering effect on the amyloid–amyloid elongation surface. Additionally, the complex protected neuronal cells from amyloid toxicity.     

The Interplay between Angiopoietin-Like Proteins and Adipose Tissue: Another Piece of the Relationship between Adiposopathy and Cardiometabolic Diseases?

Angiopoietin-like proteins, namely ANGPTL3-4-8, are known as regulators of lipid metabolism. However, recent evidence points towards their involvement in the regulation of adipose tissue function. Alteration of adipose tissue functions (also called adiposopathy) is considered the main inducer of metabolic syndrome (MS) and its related complications. In this review, we intended to analyze available evidence derived from experimental and human investigations highlighting the contribution of ANGPTLs in the regulation of adipocyte metabolism, as well as their potential role in common cardiometabolic alterations associated with adiposopathy. We finally propose a model of ANGPTLs-based adipose tissue dysfunction, possibly linking abnormalities in the angiopoietins to the induction of adiposopathy and its related disorders.     

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.     

TLR7/8 in the Pathogenesis of Parkinson’s Disease

Neuroinflammation and autoimmune mechanisms have a key part in the pathogenesis of Parkinson’s disease (PD). Therefore, we evaluated the role of Toll-like receptors (TLRs) as a link between inflammation and autoimmunity in PD. An in vivo model of PD was performed by administration of 1-metil 4-fenil 1,2,3,6-tetraidro-piridina (MPTP) at the dose of 20 mg/kg every 2 h for a total administration of 80/kg, both in single Knock Out (KO) mice for TLR7, TLR 8, and TLR9 and in double KO mice for TLR 7/8^-/-. All animals were compared with WT animals used as a control group. All animals were sacrificed after 7 days form the first administration of MPTP. The genetic absence of TLR 7 and 8 modified the PD pathway, increasing the immunoreactivity for TH and DAT compared to PD groups and decreasing microglia and astrocytes activation. Moreover, the deletion of TLR7 and TLR8 significantly reduced T-cell infiltration in the substantia nigra and lymph nodes, suggesting a reduction of T-cell activation. Therefore, our result highlights a possibility that an immunotherapy approach, by using a dual antagonist of TLR 7 and 8, could be considered as a possible target to develop new therapies for Parkinson diseases.