Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Impact of Sample Pretreatment and Extraction Methods on the Bioactive Compounds of Sugar Beet (Beta vulgaris L.) Leaves.

To find the most optimal green valorization process of food by-products, sugar beet (Beta vulgaris L.) leaves (SBLs) were freeze-dried and ground with/without liquid nitrogen (LN), as a simple sample pretreatment method, before ultrasound-assisted extraction (UAE) of polyphenols. First, the water activity, proximate composition, amino acid (AA) and fatty acid (FA) profiles, and polyphenol oxidase (PPO) activity of dried and fresh SBLs were evaluated. Then, conventional extraction (CE) and UAE of polyphenols from SBLs using water/EtOH:water 14:6 (v/v) as extracting solvents were performed to determine the individual and combined effects of the sample preparation method and UAE. In all the freeze-dried samples, the specific activity of PPO decreased significantly (p ≤ 0.05). Freeze-drying significantly increased (p ≤ 0.05) the fiber and essential FA contents of SBLs. The FA profile of SBLs revealed that they are rich sources of oleic, linoleic, and α-linolenic acids. Although freeze-drying changed the contents of most AAs insignificantly, lysine increased significantly from 7.06 ± 0.46% to 8.32 ± 0.38%. The aqueous UAE of the freeze-dried samples without LN pretreatment yielded the most optimal total phenolic content (TPC) (69.44 ± 0.15 mg gallic acid equivalent/g dry matter (mg GAE/g DM)) and excellent antioxidant activities. Thus, combining freeze-drying with the aqueous UAE method could be proposed as a sustainable strategy for extracting bioactive compounds from food by-products.

Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants.

A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 microm, 300A, 150 x 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including beta-CN(I) were detected and separated simultaneously in less then 40 min, with the exception of alpha(S1)-CN(B) and CN(C) variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R(2)>0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.