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Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 3' of mRNA and drive the release of the polypeptide chain. However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

In hemophilia A (HA), F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favour inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTCs) may contribute to immune tolerance by producing full-length (FL) proteins through the insertion of amino acid subset(s). To quantitatively evaluate in vitro the readthrough output, we developed a very sensitive luciferase-based system to detect very low FL-FVIII synthesis from a wide panel (n=45; ~60% patients with PTCs) of F8 nonsense variants. PTCs not associated with inhibitor displayed higher readthrough-driven expression levels than inhibitor-associated PTCs, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six HA patients with PTCs, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTCs not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (four out of 57). These original findings into HA molecular genetics, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favour PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.

The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl.The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype.Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2.Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (tThe p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 30 of mRNA and drive the release of the polypeptide chain.However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.

Pre-peptide regions of secreted proteins display wide sequence variability, even among highly homologous proteins such as coagulation factors, and are intracellularly removed, thus potentially favoring secretion of wild-type proteins upon suppression of nonsense mutations (translational readthrough). As models we selected F9 nonsense mutations with readthrough-favorable features affecting the pre-peptide and pro-peptide regions of coagulation factor IX (FIX), which cause hemophilia B (HB). Only the p.Gly21Ter (c.61G > T) in the variable pre-peptide hydrophobic core significantly responded (secretion, 4.1 ± 0.5% of wild-type; coagulant activity, 4.0 ± 0.3%) to the readthrough-inducer geneticin. Strikingly, for the p.Gly21Ter mutation, the resulting specific coagulant activity (0.96 ± 0.11) was compatible with normal function, thus suggesting secretion of FIX with wild-type features upon readthrough and removal of pre-peptide. Expression of the predicted readthrough-deriving missense variants (Gly21Trp/Cys/Arg) revealed a preserved specific activity (ranging from 0.84 to 0.98), thus supporting our observation. Conversely, rescue of the p.Cys28Ter (c.84T > A) and p.Lys45Ter (c.133A > T) was prevented by constraints of adjacent cleavage sites, a finding consistent with the association of most missense mutations affecting these regions with severe or moderate HB. Overall, our data indicate that suppression of nonsense mutations in the pre-peptide core preserves mature protein features, thus making this class of mutations preferred candidates for therapeutic readthrough.

In this study the antioxidant composition of fresh and stored strawberries and the bioavailability of the main strawberry bioactive compounds were determined in humans. In addition we have investigated plasma total antioxidant capacity. On 13 healthy volunteers, blood samples were collected before and after acute ingestion of fresh and stored strawberries, 300g respectively. Ferric Reducing Antioxidant Power (FRAP) values after consumption of fresh and stored strawberries showed a significant increase (P < 0.05) at 5 and 8 hours time interval, while significantly decreased TRAP (Total Radical-Trapping Antioxidant Parameter) values (P < 0.05) were found at 8 hours respect to baseline after stored strawberries consumption. After consuming fresh strawberries, plasma levels of α-carotene increased significantly (P < 0.05) respect to stored ones. Moreover, consumption of fresh and stored strawberries resulted in a significant increase of vitamin C at 2, 3 and 5 hours (P < 0.05). The bioavailable amount of strawberries antioxidant compounds reflects the variations observed in fresh and stored fruits. We could summarize that the global food quality is related to both native quantity of bioactive compounds and storage treatments.

Plant-based foods (such as fruit and vegetables, wine, nuts, natural vegetable oils, and whole grains) are an important component of traditional diets in Mediterranean regions. A large, consistent body of scientific evidence demonstrates that diets rich in plant foods provide protection against degenerative diseases; however, despite the consensus of the evidence about the health effect of plant foods, it is unclear which components of plant-based foods are protective and what their mechanism of action is. One of the hypotheses postulated to explain the protective effect of plant food, the antioxidant hypothesis, is based on their high content of bioactive molecules. Recent evidence suggests that it is the variegate composition of the plant food, an optimal mixture of different antioxidants endowed with complementary mechanism of action and different redox potential, which is at the basis of their effect on health. The global antioxidant efficiency of complex matrixes can be assessed by measuring their total antioxidant capacity (TAC) representing the result of variables such as redox potentials of the compounds present in the matrix and their cumulative and synergistic interaction. In the last years different databases for TAC of plant foods have been developed. Results suggest that coffee might represent a potential contributor to dietary antioxidant intake. In this contribution after describing the main contributors to dietary TAC for different plant food group, we will discuss the potentiality of coffee as a source of "ready to drink" reducing equivalents.

The antioxidant properties of dietary phenolics are believed to be reduced in vivo because of their affinity for proteins. In this study we assessed the bioavailability of phenolics and the in vivo plasma antioxidant capacity after the consumption of blueberries (Vaccinium corymbosum L.) with and without milk. In a crossover design, 11 healthy human volunteers consumed either (a) 200 g of blueberries plus 200 ml of water or (b) 200 g of blueberries plus 200 ml of whole milk. Venous samples were collected at baseline and at 1, 2, and 5 h postconsumption. Ingestion of blueberries increased plasma levels of reducing and chain-breaking potential (+6.1%, p

International audience; The present study investigated the effect of the consumption of two cheese varieties differing for fat quality on blood lipid profile and redox status biomarkers in 30 selected healthy volunteers, consuming either the experimental cheese (from milk produced by cows fed a grass and maize silage based diet with 5% of linseed oil added) or the control cheese (from normal cows' milk) for 4 weeks according to a crossover design. The experimental cheese had a lower content of medium-chain saturated fatty acids and a higher content of stearic acid and polyunsaturated fatty acids; its consumption led to higher levels of vitamins C and E and stearic acid in blood, while myristic acid and oxidized low-density lipoprotein concentrations were significantly lower. As myristic acid and oxidized low-density lipoprotein are highly correlated with increased atherogenic risk and vitamins C and E with antioxidant activity, the enrichment of cows' diet with linseed oil could provide a dietary option to prevent cardiovascular diseases risk.

The aim of the study was to assess the effect of drinking rooibos tea (Aspalathus linearis) on total antioxidant capacity (TAC), lipid triacylglycerols, cholesterol and glycaemia plasma levels in humans. In vitro, unfermented rooibos tea displayed a 28% higher value of TRAP than did the fermented beverage. An acute intervention study, cross-over design, was performed, with 15 healthy volunteers who consumed 500ml of either water, unfermented or fermented rooibos teas. Plasma antioxidant capacity increased significantly with both teas, reaching a peak at 1h post-consumption (+6.6%, p

In a randomized cross-over study, 15 healthy volunteers consumed 500 mL of green tea (GTFT) with different solid contents (1.4, 1.6, 1.8 and 2.0 g/L) to induce a dose-response effect on plasma antioxidant capacity. Ingestion of GTFT 2.0 g/L significantly increased plasma reducing power (ferric reducing antioxidant power, FRAP) at 1 h (+2.9%; p