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HPLC–MS applications in the agrifood sector are among the fastest developing fields in science and industry. The present tutorial mini-review briefly describes this analytical methodology: HPLC, UHPLC, nano-HPLC on one hand, mass spectrometry (MS) and tandem mass spectrometry (MS/MS) on the other hand. Analytical results are grouped together based on the type of chemicals analyzed (lipids, carbohydrates, glycoproteins, vitamins, flavonoids, mycotoxins, pesticides, allergens and food additives). Results are also shown for various types of food (ham, cheese, milk, cereals, olive oil and wines). Although it is not an exhaustive list, it illustrates the main current directions of applications. Finally, one of the most important features, the characterization of food quality (including problems of authentication and adulteration) is discussed, together with a future outlook on future directions.

The primary structure of four α(s2)-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkey's α(s2)-CN (GenBank Acc. No. CAV00691; M(r) 26,028 Da) as reference. Proteins, with experimentally measured M(r) of 25,429, 21,939, 25,203 and 21,713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, α-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.

Two coeluting components, with experimentally measured M(r) values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey beta-CNs and their sequences characterized completely, using the two known beta-CNs from mare as references. The two donkey beta-CNs, showing a mass difference of 923 Da, differ by the presence of the domain E(27)SITHINK(34) in the full-length component (M(r) 25529 Da). In comparison with the mare's beta-CNs used as reference, they present nine amino acid substitutions: L--S(37), R--H(52), S--N(81), P--V(84), L--V(91), R--Q(203), P--L/I(206), L--F(210) and A--P(219). Together, these substitutions account for the increase of 18 Da in the M(r) of the donkey beta-CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's beta-CNs, the full-length (226 amino acids) beta-CN was termed variant A, whereas the shorter (218 amino acids) beta-CN was termed variant A(Delta5).

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brain-extracted myelin were produced. The MyVes have an average diameter of 100–150 ​nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases., Graphical abstract Image 1, Highlights • Bio-fabrication of brain tissue derived nanovesicles: myelin nanovesicles. • Myelin nanovesicles contain the main proteins of the myelin sheath (myelin basic protein and myelin proteolipid protein). • Myelin nanovesicles can lade a drug/molecule and cross a blood–brain barrier model. • Myelin nanovesicles target white matter and microglia cells.

Type 2 diabetes mellitus (T2DM) is an important metabolic disorder for which there is an urgent need for new antidiabetic drugs. α-Glucosidase inhibition is an established protocol for T2DM therapy. Because hyperglycemia causes oxidative tissue damage, the development of agents with both α-glucosidase inhibition and antioxidant activity from natural or natural-derived polyphenols such derivatives of rosmarinic acid (RA) represents an attractive therapeutic option. We report a study on amides 1-10 derived from RA and their evaluation for yeast α-glucosidase inhibition and antioxidant activity (DPPH and ORAC tests). All amides showed higher inhibitory activity than that of RA, were by far more potent than the antidiabetic drug acarbose, and proved to be effective antioxidants. A molecular docking study displayed significant binding interactions of RA amides with the active site of α-glucosidase. This in silico optimization study led to the design and synthesis of amides 9 (IC 50 = 42.3 μM) and 10 (IC 50 = 35.2 μM), showing the most potent α-glucosidase inhibition and good antioxidative properties. A kinetic study showed that 10 acts as a mixed type inhibitor.

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R ® ) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and β-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 μg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 μg/ml, while TRE 20 μg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 μg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 μg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 μg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and β-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.

Aims: Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system. Methods and Results: We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253.7 to 15.8 mg ml(-1). We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31.7 mg ml(-1)) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited. Conclusions: The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9-41 of a beta-thymosin of P. lividus. Significance and Impact of the Study: The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms