Cellular uptakes, biostabilities and anti-miR-210 activities of chiral arginine-PNAs in leukaemic K562 cells

A series of 18‐mer peptide nucleic acids (PNAs) targeted against micro‐RNA miR‐210 was synthesised and tested in a cellular system. Unmodified PNAs, R₈‐conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2‐modified (R) or C5‐modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT‐PCR on K562 chronic myelogenous leukaemic cells indicated that arginine‐conjugated and backbone‐modified PNAs display good cellular uptake, with best performances for the C2‐modified series. Resistance to enzymatic degradation was found to be higher for the backbone‐modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR‐210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin‐treated cells. Interestingly, the anti‐miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone‐modified PNAs as anti‐miR agents. The results clearly indicate that backbone‐modified PNAs are good candidates for the development of very efficient drugs based on anti‐miR activity, due to their enhanced bioavailabilities, and that overall anti‐miR performance is a combination of cellular uptake and RNA binding.     

HCB, p,p'-DDE and PCB ontogenetic transfer and magnification in bluefin tuna (Thunnus thynnus) from the Mediterranean Sea

The bluefin tuna, Thunnus thynnus (Linnaeus 1758), is biologically and economically important in the Atlantic--Mediterranean ecosystems. Bluefin tuna feed on diverse food items depending on their age, thus they occupy different trophic levels during their lifespan. Hexachlorobenzene (HCB), p,p'-DDE and polychlorinated biphenyls (PCBs) are well-known persistent organic pollutants (POPs) in the Mediterranean basin. The relationship between stable isotopes of nitrogen (N) and the POP residue levels in tissues has recently increased knowledge on the link between the trophic levels and the contaminant accumulation. Trophic levels were estimated by using 15N/14N ratio (delta15N) and HCB, p,p'-DDE, and forty-three PCBs were quantified in bluefin tuna from the southern Tyrrhenian Sea. Results showed that changes in PCB and p,p'-DDE concentrations were a function of size and trophic level, while no correlations were observed for HCB. Apart from HCB and PCB nos. 101, 207, 95, 158, and 60 + 56, which did not show any significant increase per trophic level, the other PCBs and the p,p'-DDE increased significantly. The ontogenetic magnification factor of PCBs was 6.6 +/- 0.5, which was significantly (12 times) higher (p < 0.05) than the values found for p,p'-DDE (1.4) and HCB (1.4).     

Total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in Italy assessed by three different in vitro assays

With the aim to expand the Italian total antioxidant capacity (TAC) database, the TAC values of 11 spices, 5 dried fruits, 7 sweets, 18 cereal products, 5 pulses, and 6 nuts were determined using three different assays and considering the contribution of bound antioxidant compounds in fiber-rich foods (i. e. cereals, legumes, and nuts). Among spices, saffron displayed the highest antioxidant capacity, whereas among dried fruits, prune exhibited the highest value. The TAC values of all the chocolates analyzed were far higher than the other sweet extracts measured. Among cereal products, whole meal buckwheat and wheat bran had the greatest TAC. Among pulses and nuts, broad bean, lentil and walnuts had the highest antioxidant capacity, whereas chickpeas, pine nuts and peanuts were less effective. The contribution of bound phytochemicals to the overall TAC was relevant in cereals as well as in nuts and pulses. The complete TAC database could be utilized to properly investigate the role of dietary antioxidants in disease prevention.     

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening

Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe's milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses.     

Intra-Laboratory Evaluation of DNA Extraction Methods and Assessment of a Droplet Digital PCR for the Detection of Xanthomonas citri pv. citri on Different Citrus Species

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method.     

Evaluating the Efficiency of Chitosan-Graphene Oxide Hybrid Hydrogels as Drug Delivery Systems

With the objective of creating an electro-responsive and antimicrobial device suitable as delivery system for Rose Bengal (RB) to the skin, a hybrid hydrogel combining Chitosan (CS) and Graphene Oxide (GO) are designed, serving as functional polymer support and active filling element, respectively. The hybrid system, synthesized using tripolyphosphate as a crosslinker via ionic gelation, shows a uniform and homogeneous surface, as verified by SEM investigations, high biocompatibility when tested on human fibroblast lung cells MRC-5 cells, and biodegradability in phosphate buffered medium at physiological pH. Drug loading and release experiments, extensively analyzed using suitable mathematical modeling, shows the enhancement of the binding efficiency conferred by GO (534 and 979 mg g⁻¹ for blank and hybrid hydrogels, respectively) and an electro-responsive behavior (maximum BR release of 36 and 23% at 0 and 12 V, respectively). Additionally, hybrid hydrogel is found to prevent the adhesion of methicillin-resistant Staphylococcus aureus and to kill the bacterial cells by taking advantage of the sustained release of the antimicrobial RB.     

Biotransamination of Furan-Based Aldehydes with Isopropylamine: Enzyme Screening and pH Influence

Furan-based amines are highly valuable compounds which can be directly obtained via reductive amination from easily accessible furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). Herein the biocatalytic amination of these carbonyl derivatives is disclosed using amine transaminases (ATAs) and isopropylamine (IPA) as amine donors. Among the different biocatalysts tested, the ones from Chromobacterium violaceum (Cv-TA), Arthrobacter citreus (ArS-TA), and variants from Arthrobacter sp. (ArRmut11-TA) and Vibrio fluvialis (Vf-mut-TA), afforded high levels of product formation (>80 %) at 100–200 mM aldehyde concentration. The transformations were studied in terms of enzyme and IPA loading. The pH influence was found as a key factor and attributed to the imine/aldehyde equilibrium that can arise from the high reactivity of the carbonyl substrates with a nucleophilic amine such as IPA.     

Prevention of Swelling Phenomenon of Alginate Beads To Improve the Stability and Recyclability of Encapsulated Horse Liver Alcohol Dehydrogenase

Horse Liver Alcohol Dehydrogenase (HLADH) has been immobilized on calcium-alginate beads and used for both oxidation and reduction reactions. To avoid swelling of the beads and their subsequent breakage, calcium ions were added to both reaction and storage solutions, allowing the beads to maintain the initial structural features. The techniques used for this purpose revealed that 2 mM Ca²⁺ is the optimal concentration, which does not significantly change the weight of the beads, the amount of water in them, and their external and internal structure. The optimized experimental procedure has been used to verify the properties of the enzyme in terms of reusability, storage, and thermal stability. The addition of calcium ions allows the enzyme to retain more than 80 % of its initial activity for fourteen cycles and approximately 50 % at the twentieth cycle. Moreover, when the biocatalyst has been stored in a buffer solution containing 2 mM Ca²⁺, the retention of enzyme activity after 30 days was 100 %, compared to that measured before incubation. The encapsulated enzyme exhibits greater thermal stability than free HLADH up to at least 60 °C, preventing dimer dissociation into the two subunits.