Combat phytopathogenic bacteria employing Argirium-SUNCs: limits and perspectives.

Bacterial plant diseases are difficult to control as the durability of deployed control measures is thwarted by continuous and rapid changing of bacterial populations. Although application of copper compounds to plants is the most widespread and inexpensive control measure, it is often partially efficacious for the frequent appearance of copper-resistant bacterial strains and it is raising concerns for the harmful effects of copper on environment and human health. Consequently, European Community included copper compounds in the list of substances candidates for substitution. Nanotechnologies and the application of nanoparticles seem to respond to the need to find new very effective and durable measures. We believe that Argirium-SUNCs®, silver ultra nanoclusters with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+), represent a valid candidate as a nano-bactericide in the control of plant bacterial diseases. Respect to the many silver nanoparticles described in the literature, Argirium-SUNCs have many strengths due to the reproducibility of the synthesis method, the purity and the stability of the preparation, the very strong (less than 1 ppm) antimicrobial, and anti-biofilm activities. In this mini-review, we provide information on this nanomaterial and on the possible application in agriculture. KEY POINTS: • Argirium-SUNCs have strong antimicrobial activities against phytopathogenic bacteria. • Argirium-SUNCs are a possible plant protection product. • Argirium-SUNCs protect tomato plants against bacterial speck disease.

Silver nanoclusters with Ag2+/3+ oxidative states are a new highly effective tool against phytopathogenic bacteria.

The main measure worldwide adopted to manage plant bacterial diseases is based on the application of copper compounds, which are often partially efficacious for the frequent appearance of copper-resistant bacterial strains and have raised concerns for their toxicity to the environment and humans. Therefore, there is an increasing need to develop new environmentally friendly, efficient, and reliable strategies for controlling plant bacterial diseases, and among them, the use of nanoparticles seems promising. The present study aimed to evaluate the feasibility of protecting plants against attacks of gram-negative and gram-positive phytopathogenic bacteria by using electrochemically synthesized silver ultra nanoclusters (ARGIRIUM­SUNCs®) with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+). ARGIRIUM­SUNCs strongly inhibited the in vitro growth (effective concentration, EC50, less than 1 ppm) and biofilm formation of Pseudomonas syringae pv. tomato and of quarantine bacteria Xanthomonas vesicatoria, Xylella fastidiosa subsp. pauca, and Clavibacter michiganensis subsp. michiganensis. In addition, treatments with ARGIRIUM­SUNCs also provoked the eradication of biofilm for P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis. Treatment of tomato plants via root absorption with ARGIRIUM­SUNCs (10 ppm) is not phytotoxic and protected (80%) the plants against P. syringae pv. tomato attacks. ARGIRIUM­SUNCs at low doses induced hormetic effects on P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis as well as on tomato root growth. The use of ARGIRIUM­SUNCs in protecting plants against phytopathogenic bacteria is a possible alternative control measure. KEY POINTS: • ARGIRIUM­SUNC has strong antimicrobial activities against phytopathogenic bacteria; • ARGIRIUM­SUNC inhibits biofilm formation at low doses; • ARGIRIUM­SUNC protects tomato plants against bacterial speck disease.

Structure and Properties of Electrochemically Synthesized Silver Nanoparticles in Aqueous Solution by High-Resolution Techniques.

The aim of this work was to deeply investigate the structure and properties of electrochemically synthesized silver nanoparticles (AgNPs) through high-resolution techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta Potential measurements, and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Strong brightness, tendency to generate nanoclusters containing an odd number of atoms, and absence of the free silver ions in solution were observed. The research also highlighted that the chemical and physical properties of the AgNPs seemed to be related to their peculiar oxidative state as suggested by X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRPD) analyses. Finally, the MTT assay tested the low cytotoxicity of the investigated AgNPs.

Identification and characterization of the α-CA in the outer membrane vesicles produced by Helicobacter pylori.

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and ß-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.

Effect of membrane depolarization against Aspergillus niger GM31 resistant by ultra nanoclusters characterized by Ag2+ and Ag3+ oxidation state.

To date, the impossibility of treating resistant forms of bacteria and fungi (AMR) with traditional drugs is a cause for global alarm. We have made the green synthesis of Argirium silver ultra nanoclusters (Argirium-SUNCs) very effective against resistant bacteria (< 1 ppm) and mature biofilm (0.6 ppm). In vitro and preclinical tests indicate that SUNCs are approximately 10 times less toxic in human cells than bacteria. Unique chemical-physical characteristics such as particle size < 2 nm, a core composed of Ag0, and a shell of Ag +, Ag2+ , Ag3+ never observed before in stable form in ultra pure water, explain their remarkable redox properties Otto Cars (Lancet Glob. Health 9:6, 2021). Here we show that Argirium-SUNCs have strong antimicrobial properties also against resistant Aspergillus niger GM31 mycelia and spore inactivation (0.6 ppm). The membrane depolarization is a primary target leading to cell death as already observed in bacteria. Being effective against both bacteria and fungi Argirium-SUNCs represent a completely different tool for the treatment of infectious diseases.

The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death.

This work highlights how our silver ultra nanoclusters (ARGIRIUM-SUNc) hand-made synthesized, are very useful as a bactericide and anti-biofilm agent. The Argirium-SUNc effective antibacterial concentrations are very low (< 1 ppm) as compared to the corresponding values reported in the literature. Different bacterial defense mechanisms are observed dependent on ARGIRIUM-SUNc concentrations. Biochemical investigations (volatilome) have been performed to understand the pathways involved in cell death. By using fluorescence techniques and cell viability measurements we show, for the first time, that membrane depolarization and calcium intracellular level are both primary events in bacteria death. The ARGIRIUM-SUNc determined eradication of different biofilm at a concentration as low as 0.6 ppm. This suggests that the effect of the nanoparticles follows a common mechanism in different bacteria. It is highly probable that the chemical constitution of the crosslinks could be a key target in the disrupting mechanism of our nanoparticles. Since the biofilms and their constituents are essential for bacterial survival in contact with humans, the silver nanoparticles represent a logical target for new antibacterial treatments.

Antimicrobial and Antibiofilm Activities of New Synthesized Silver Ultra-NanoClusters (SUNCs) Against Helicobacter pylori.

Helicobacter pylori colonizes approximately 50% of the world's population, and it is the cause of chronic gastritis, peptic ulcer disease, and gastric cancer. The increase of antibiotic resistance is one of the biggest challenges of our century due to its constant increase. In order to identify an alternative or adjuvant strategy to the standard antibiotic therapy, the in vitro activity of newly synthesized Silver Ultra-NanoClusters (SUNCs), characterized by an average size inferior to 5 nm, against clinical strains of H. pylori, with different antibiotic susceptibilities, was evaluated in this study. MICs and MBCs were determined by the broth microdilution method, whereas the effect of drug combinations was determined by the checkerboard assay. The Minimum Biofilm Eradication Concentration (MBEC) was measured using AlamarBlue (AB) assay and colony-forming unit (CFU) counts. The cytotoxicity was evaluated by performing the MTT assay on the AGS cell line. The inhibitory activity was expressed in terms of bacteriostatic and bactericidal potential, with MIC50, MIC90, and MBC50 of 0.33 mg/L against planktonic H. pylori strains. Using the fractional inhibitory concentration index (FICI), SUNCs showed potential synergism with metronidazole and clarithromycin. The biofilm eradication was obtained after treatment with 2×, 3×, and 4× MIC values. Moreover, SUNCs showed low toxicity on human cells and were effective in eradicating a mature biofilm produced by H. pylori. The data presented in this study demonstrate that SUNCs could represent a novel strategy for the treatment of H. pylori infections either alone or in combination with metronidazole.

Proteomic Investigation of Malignant Major Salivary Gland Tumors.

The purpose of this study was to define the proteome profile of fine needle aspiration (FNA) samples of malignant major salivary gland tumors (MSGT) compared to benign counterparts, and to evaluate potential clinical correlations and future applications. Patients affected by MSGT (n = 20), pleomorphic adenoma (PA) (n = 37) and Warthin's tumor (WT) (n = 14) were enrolled. Demographic, clinical and histopathological data were registered for all patients. FNA samples were processed to obtain the protein extracts. Protein separation was obtained by two-dimensional electrophoresis (2-DE) and proteins were identified by mass spectrometry. Western blot analysis was performed to validate the 2-DE results. Statistical differences between groups were calculated by the Mann-Whitney U test for non-normal data. Spearman's rank correlation coefficient was calculated to evaluate correlations among suggested protein biomarkers and clinical parameters. Twelve and 27 differentially expressed spots were found for MSGT versus PA and MSGT versus WT, respectively. Among these, annexin-5, cofilin-1, peptidyl-prolyl-cis-trans-isomerase-A and F-actin-capping-alpha-1 were able to differentiate MSGT from PA, WT, and healthy samples. Moreover, STRING analysis suggested cofilin-1 as a key node of protein interactions. Some of the overexpressed proteins are related to some clinical factors of our cohort, such as survival and outcome. Our results suggest potential protein biomarkers of MSGT, which could allow for more appropriate treatment plans, as well as shedding light on the molecular pathways involved.

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the "mitochondrial dimethylation-TAILS" strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 - MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

Electrochemically Synthesized Silver Nanoparticles Are Active Against Planktonic and Biofilm Cells of Pseudomonas aeruginosa and Other Cystic Fibrosis-Associated Bacterial Pathogens.

A novel, electrochemically synthesized, silver nanoparticles (AgNPs) formulation was evaluated in vitro against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Staphylococcus aureus strains from cystic fibrosis (CF) patients. AgNPs were particularly active against P. aeruginosa and B. cepacia planktonic cells (median MIC: 1.06 and 2.12 µg/ml, respectively) by a rapid, bactericidal and concentration-dependent effect. AgNPs showed to be particularly effective against P. aeruginosa and S. aureus biofilm causing a viability reduction ranging from 50% (1×MIC) to >99.9% (4×MIC). Electron microscopy showed that AgNPs deconstruct extracellular matrix of P. aeruginosa biofilm, and accumulate at the cell surface causing cell death secondary to membrane damage. Compared to Tobramycin, AgNPs showed comparable, or even better, activity against planktonic and biofilm P. aeruginosa cells. AgNPs at concentrations effective against B. cepacia and P. aeruginosa were not toxic to G. mellonella larvae. Our silver-based formulation might be an alternative to antibiotics in CF patients. Further in vitro and in vivo studies are warranted to confirm this therapeutic potential.

Proteomic Characterization of a New asymmetric Cellulose Triacetate Membrane for Hemodialysis.

PURPOSE: The artificial membrane inside the haemodialyzer is the main determinant of the quality and success of haemodialysis therapy. The performances of haemodialysis membranes are highly influenced by the interactions with plasma proteins, which in turn are related to the physical and chemical characteristics of the membrane material. The present cross-over study is aimed to analyse the haemodialysis performance of a newly developed asymmetric cellulose triacetate membrane (ATA) in comparison to the conventional parent symmetric polymer (CTA). EXPERIMENTAL DESIGN: In four chronic non diabetic haemodialysis patients, the protein constituents of the adsorbed material from the filters after the haemodialysis session, and the proteins recovered in the ultrafiltrate during the session, are identified using a bottom-up shotgun proteomics approach. RESULTS: The ATA membrane shows a lower protein adsorption rate and a lower mass distribution pattern of the proteinaceous material. CONCLUSIONS AND CLINICAL RELEVANCE: By highlighting the differences between the two haemodialysis filters in terms of adsorbed proteins and flow through, it is demonstrated the higher biocompatibility of the novel ATA membrane, that fulfils the indications for the development of more performant membranes and may represent a step forward for the treatment of patients on chronic haemodialysis.

Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90-231, as possible mechanism of its neurotoxic effects.

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90-231 (incubated for 1 h at 53 degrees C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants.

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP(ARQ) [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP(ARR) [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP(ARR) and PrP(ARQ) variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP(ARR) form was more toxic than the scrapie susceptible PrP(ARQ) variant. Moreover, the alpha-helical conformation of PrP(ARR) was less stable than that of PrP(ARQ) and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP(ARQ) variant displays a higher propensity to form large aggregates than PrP(ARR). Interestingly, in the presence of small amounts of PrP(ARR), PrP(ARQ) aggregability was reduced to levels similar to that of PrP(ARR). Thus, in contrast to PrP(ARR) toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP(ARQ) that allows the formation of large amyloid fibrils.

Amino-terminally truncated prion protein PrP90-231 induces microglial activation in vitro.

The conversion of the prion protein (PrP) into a protease-resistant isoform (PrP(Res)) is considered the pathogenic event responsible for prion encephalopathies. Microglia activation accompanies PrP(Res) deposition representing an early event in the progression of these diseases. It is now believed that microglial cells play a worsening, if not causative, role in prion-induced neuronal death, through the release of proinflammatory and neurotoxic molecules. Indeed, in vitro observations have demonstrated that PrP(Res) and the synthetic prion fragment PrP106-126 induce neuronal death by activating microglial to migrate in the lesion area and secrete cytokines. Recently, we and others have demonstrated that the recombinant peptide, corresponding to the protease-resistant portion of PrP encompassing the amino acids 90-231 (PrP90-231), when beta-structured, is toxic for neuronal cells, in vitro. Here we report that PrP90-231 induces activation of N9 microglial cells, characterized by cell proliferation arrest and increased secretion of different cytokines (RANTES, GCSF, and IL-12). Moreover, the treatment of N9 cells with PrP90-231 elicited inducible nitric oxide synthase (i-NOS) expression, nitric oxide release, and a delayed (15 min to 1 h of treatment) extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation/activation. Although ERK1/2 is known to regulate proliferative and differentiative events, we show that its blockade, using the specific MEK inhibitor PD98059, did not prevent PrP90-231-induced inhibition of N9 cell proliferation. To our knowledge, this is the first evidence that a recombinant PrP(Res)-like peptide elicits microglial activation in vitro, thus representing a potentially important tool to develop possible therapeutic strategies to target prion-induced brain inflammation.

Characterization of the proapoptotic intracellular mechanisms induced by a toxic conformer of the recombinant human prion protein fragment 90-231.

Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly alpha-structured isoform (PrPC) to a prevalent beta-sheet-containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90-231 of human prion protein (hPrP90-231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90-231 into a beta-sheet-rich isoform, displaying a temperature- and time-dependent conversion into oligomeric structures that share some physico-chemical characteristics with brain PrPSc. SH-SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90-231 in its different structural conformations. We demonstrated that hPrP90-231 in beta-conformation, but not when alpha-structured, powerfully affected the survival of these cells. hPrP90-231 beta-structured caused DNA fragmentation and a significant increase in caspase-3 proteolytic activity (maximal effects+170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of beta-hPrP90-231-dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90-231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90-231 elicits proapoptotic activity when in beta-sheet-rich conformation and that this effect is mediated by p38 and caspase-3 activation.

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics.

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes. Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 microM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs. The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.

Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity.

Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE.

Rosuvastatin effect on intima media thickness in adult vs elderly patients.

The benefits of cardiovascular therapies such as statins for the treatment of atherosclerosis have been well documented. Many studies have demonstrated important benefits in patients with asymptomatic carotid atherosclerosis. We have evaluated the effect of low dose of rosuvastatin on asymptomatic carotid atherosclerosis in elderly versus adult subjects. Among 640 participants in the Asymptomatic Carotid Atherosclerotic Disease In Manfredonia Study (ACADIM Study) forty-five patients (21 adults, 24 elderly) with hypercholesterolemia and asymptomatic carotid atherosclerosis on baseline carotid ultrasound investigation (CUI) were examined with repeat CUI after one treatment year with rosuvastatin (ROS) (10 mg/day). Total and low density lipoprotein cholesterol decreased significantly (p<0.001) while high density lipoprotein cholesterol increased significantly (p<0.001) during the intervention. Mean decrease in carotid intima media thickness (CIMT) of the right and left common carotid arteries were higher in adult versus elderly subjects (p<0.04 for each), even if in both group there was a significant regression in carotid atherosclerosis respect to baseline values (P<0.001). These results confirm the reduction in IMT of the CCAs in response to ROS at a low dose in a one-year treatment period, even if in elderly subjects this effect is lower respect to adult. The treatment of asymptomatic carotid atherosclerosis defined by CIMT started in the adult age is more effective.

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP90₋231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP90₋231, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90₋231 bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP90₋231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90₋231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP90₋231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.