A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ORF Virus through the Whole Genome Sequencing of the First Italian Strains.

Orf virus (ORFV) is distributed worldwide and is the causative agent of contagious ecthyma that mainly occurs in sheep and goats. This disease was reported for the first time at the end of 18th century in Europe but very little is currently known about the temporal and geographic origins of this virus. In the present study, the use of new Italian whole genomes allowed for better inference on the evolutionary history of ORFV. In accordance with previous studies, two genome types (S and G) were described for infection of sheep and goats, respectively. These two well-differentiated groups of genomes originated for evolutive convergence in the late 1800s in two different areas of the world (Europe for S type and Asia for G type), but it was only in the early 1900s that the effective size of ORFV increased among hosts and the virus spread across the whole European continent. The Italian strains which were sequenced in the present study were isolated on the Mediterranean island of Sardinian and showed to be exclusive to this geographic area. One of them is likely representative of the early European forms of ORFV which infected sheep and became extinct about one century ago. Such an ancient Sardinian strain may have reached the island simple by chance, where it quickly adapted to the new habitat.

Molecular Insights into the Genetic Variability of ORF Virus in a Mediterranean Region (Sardinia, Italy).

Orf virus (ORFV) represents the causative agent of contagious ecthyma, clinically characterized by mild papular and pustular to severe proliferative lesions, mainly occurring in sheep and goats. In order to provide hints on the evolutionary history of this virus, we carried out a study aimed to assess the genetic variation of ORFV in Sardinia that hosts a large affected small ruminant population. We also found a high worldwide mutational viral evolutionary rate, which resulted, in turn, higher than the rate we detected for the strains isolated in Sardinia. In addition, a well-supported genetic divergence was found between the viral strains isolated from sheep and those from goats, but no relevant connection was evidenced between the severity of lesions produced by ORFV and specific polymorphic patterns in the two species of hosts. Such a finding suggests that ORFV infection-related lesions are not necessarily linked to the expression of one of the three genes here analyzed and could rather be the effect of the expression of other genes or rather represents a multifactorial character.

Prenylated Trans-Cinnamic Esters and Ethers against Clinical Fusarium spp.: Repositioning of Natural Compounds in Antimicrobial Discovery.

Onychomycosis is a common nail infection mainly caused by species belonging to the F. oxysporum, F. solani, and F. fujikuroi species complexes. The aim of this study was to evaluate the in vitro susceptibility of six representative strains of clinically relevant Fusarium spp. toward a set of natural-occurring hydroxycinnamic acids and their derivatives with the purpose to develop naturally occurring products in order to cope with emerging resistance phenomena. By introducing a prenylated chain at one of the hydroxy groups of trans-cinnamic acids 1-3, ten prenylated derivatives (coded 4-13) were preliminarily investigated in solid Fusarium minimal medium (FMM). Minimal inhibitory concentration (MIC) and lethal dose 50 (LD50) values were then determined in liquid FMM for the most active selected antifungal p-coumaric acid 3,3'-dimethyl allyl ester 13, in comparison with the conventional fungicides terbinafine (TRB) and amphotericin B (AmB), through the quantification of the fungal growth. Significant growth inhibition was observed for prenylated derivatives 4-13, evidencing ester 13 as the most active. This compound presented MIC and LD50 values (62-250 µM and 7.8-125 µM, respectively) comparable to those determined for TRB and AmB in the majority of the tested pathogenic strains. The position and size of the prenylated chain and the presence of a free phenol OH group appear crucial for the antifungal activity. This work represents the first report on the activity of prenylated cinnamic esters and ethers against clinical Fusarium spp. and opens new avenues in the development of alternative antifungal compounds based on a drug repositioning strategy.

Use of ß-cyclodextrin as enhancer of ascorbic acid rejection in permselective films for amperometric biosensor applications.

Interference rejection in amperometric biosensors can be more effective introducing some modifiers during electro-deposition of permselective film. Addition of ß-cyclodextrin (ßCD), a cyclic oligosaccharide composed of seven glucose units, to the ortho-phenylendiamine (oPD) monomer were already demonstrated to provide an enhancement in ascorbic acid (AA) rejection. Here we evaluated the improvement in permselectivity of poly-eugenol and poly-magnolol films electro-polymerized in presence of different amounts of ßCD or eugenol-ßCD inclusion complex for amperometric biosensor application. Starting from Pt-Ir wire as transducer several microsensors were covered with polymeric films doped with ßCD-based modifiers through constant potential amperometry. Characterization of modified polymers was achieved by scanning electron microscopy and permselectivity analysis. Poly-magnolol film in combination with ßCD showed a worsening in permselectivity compared to poly-magnolol alone. In contrast, the introduction of ßCD-based modifier enhanced the interference rejection toward the archetypal interferent AA, while slightly affecting permeability toward H2O2 compared to the poly-eugenol without modifier. The AA rejection seems to be influenced by the availability of ßCD cavity as well as film performance due to concentration of ßCD-Eugenol inclusion complex. A poly-eugenol film co-polymerized with 2 mM ßCD-eugenol inclusion complex showed a permselectivity equal to poly-orthophenylendiamine film (PPD), with a lower permeability to AA, likely to be related with a self-blocking mechanism. Based on these results, a biosensor for glutamate was constructed with a poly-eugenol doped with ßCD-eugenol as permselective layer and its permselectivity, stability and lifetime were determined.

Low electro-synthesis potentials improve permselectivity of polymerized natural phenols in biosensor applications.

First-generation amperometric biosensors are often based on the electro-oxidation of oxidase-generated H2O2. At the applied potential used in most studies, other molecules such as ascorbic acid or dopamine can be oxidized. Phenylenediamines are commonly used to avoid this problem: when these compounds are electro-deposited onto the transducer surface in the form of poly-phenylenediamine, a highly selective membrane is formed. Although there is no evidence of toxicity of the resulting polymer, phenylenediamine monomers are considered carcinogenic. An aim of this work was to evaluate the suitability of natural phenols as non-toxic alternatives to the ortho isomer of phenylenediamine. Electrosynthesis over Pt-Ir electrodes of 2-methoxy phenols (guaiacol, eugenol and isoeugenol), and hydroxylated biphenyls (dehydrodieugenol and magnolol) was achieved. The potentials used in the present study are significantly lower than values commonly applied during electro-polymerization. Polymers were obtained by means of constant potential amperometry, instead of cyclic voltammetry, in order to achieve multiple polymerizations, hence decreasing the time of realization and variability. Permselective properties of natural phenols were significantly improved at low polymerization potentials. Among the tested compounds, isoeugenol and magnolol, polymerized respectively at +25mV and +170mV against Ag/AgCl reference electrode, proved as permselective as poly-ortho-phenylenediamine and may be considered as effective polymeric alternatives. The natural phenol-coated electrodes were stable and responsive throughout 14 days. A biosensor prototype based on acetylcholine esterase and choline oxidase was electro-coated with poly-magnolol in order to evaluate the interference-rejecting properties of the electrosynthesized film in an amperometric biosensor; a moderate decrease in ascorbic acid rejection was observed during in vitro calibration of biosensors.

Electropolymerized phenol derivatives as permselective polymers for biosensor applications.

Amperometric biosensors are often coated with a polymeric permselective film to avoid electroactive interference by reducing agents present in the target medium. Phenylenediamine and phenol monomers are commonly used to form these permselective films in the design of microsensors and biosensors. This paper aims to evaluate the permselectivity, stability and lifetime of polymers electrosynthesized using either constant potential amperometry (CPA) or cyclic voltammetry (CV) from naturally occurring phenylpropanoids in monomeric and dimeric forms (eugenol, isoeugenol, dehydrodieugenol and magnolol). Sensors were characterized by scanning electron microscopy and permselectivity analysis. Magnolol formed an electro-deposited polymer with a more defined three-dimensional texture in comparison with the other films. The phenol-derived films showed different permselectivity towards H2O2 over ascorbic acid and dopamine, likely to be related to the thickness and compactness of the polymer. The CV-derived films had a better permselectivity compared to the CPA-corresponding polymers. Based on these results, the permselectivity, stability and lifetime of a biosensor for glucose were studied when a magnolol coating was electro-deposited. The structural principles governing the permselectivity of the magnolol-derived film are suggested to be mainly related to the conformational flexibility of this monomer. Newly designed biosensors, coated with electropolymerized natural phenol derivatives, may represent promising analytical devices for different application fields.

Ceftriaxone blocks the polymerization of α-synuclein and exerts neuroprotective effects in vitro.

The ß-lactam antibiotic ceftriaxone was suggested as a therapeutic agent in several neurodegenerative disorders, either for its ability to counteract glutamate-mediated toxicity, as in cerebral ischemia, or for its ability to enhance the degradation of misfolded proteins, as in Alexander's disease. Recently, the efficacy of ceftriaxone in neuroprotection of dopaminergic neurons in a rat model of Parkinson's disease was documented. However, which characteristics of ceftriaxone mediate its therapeutic effects remains unclear. Since, at the molecular level, neuronal α-synuclein inclusions and pathological α-synuclein transmission play a leading role in initiation of Parkinson-like neurodegeneration, we thought of investigating, by circular dichroism spectroscopy, the capability of ceftriaxone to interact with α-synuclein. We found that ceftriaxone binds with good affinity to α-synuclein and blocks its in vitro polymerization. Considering this finding, we also documented that ceftriaxone exerts neuroprotective action in an in vitro model of Parkinson's disease. Our data, in addition to the findings on neuroprotective activity of ceftriaxone on Parkinson-like neurodegeneration in vivo, indicates ceftriaxone as a potential agent in treatment of Parkinson's disease.

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties.

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4-6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H2O2, MPP+ and MnCl2 were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.