HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1+ in human saliva

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.     

HS-SPME-GC×GC-qMS volatile metabolite profiling of Chrysolina herbacea frass and Mentha spp. leaves

Headspace solid-phase microextraction (HS-SPME) comprehensive two-dimensional (2D) gas chromatography combined with quadrupole-mass spectrometry (GC×GC-qMS) with dedicated comparative data elaboration was applied to separate chemical patterns arising from the interaction between some Mentha species and the herbivore Chrysolina herbacea, also known as the mint bug. Upon feeding on different Mentha species (Mentha spicata L., Mentha × piperita L. and Mentha longifolia L.), C. herbacea produced frass (faeces) which were characterized by a typical volatile fraction. HS-SPME GC×GC-qMS analysis of the complex volatile fraction of both mint leaf and C. herbacea frass was submitted to advanced fingerprinting analysis of 2D chromatographic data. 1,8-Cineole, found in the leaves of all the Mentha species examined, was oxidized, and C. herbacea frass yielded high rates of several hydroxy-1,8-cineoles, including 2α-hydroxy-, 3α-hydroxy-, 3β-hydroxy- and 9-hydroxy-1,8-cineole. Upon insect feeding, several unknown oxidized monoterpenes, a p-menthane diol and three unknown phenylpropanoids were also detected in the frass volatiles. In M. longifolia, the occurrence of the monoterpene piperitenone oxide was found to be toxic and associated with insect death. The results of this work show that high throughput techniques such as HS-SPME and GC×GC-qMS fingerprint analysis are ideal tools to analyze complex volatile matrices, and provide a sensitive method for the direct comparison and chemical visualization of plant and insect emitted volatile components.     

Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts

This study reported the antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben inflorescences. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic extracts were determined according to the Clinical Laboratory Standards Institute guidelines. Individual phenolic acids and flavonoids were detected by a high performance liquid chromatography (HPLC-DAD) method. The samples showed a comparable antimicrobial activity, although the natural site extract possessed the lower MIC values. The best activity was detected against Gram-positive bacteria, such as Staphylococcus aureus including methicillin resistant strains (MIC and MBC values ranging from 7.81 to 62.50?μg?mL(-1) and from 500 to 2000?μg?mL(-1) respectively). In contrast, a low activity was found on Gram-negative bacteria and Candida albicans. The HPLC-DAD analysis revealed ten phenolic acids and four flavonoids with a major amount of m-coumaric acid, naringin and quercetin in the natural site extract.     

Evaluation of rheological properties and swelling behaviour of sonicated scleroglucan samples

Scleroglucan is a natural polysaccharide that has been proposed for various applications. However there is no investigation on its property variations when the molecular weight of this polymer is reduced. Scleroglucan was sonicated at two different polymer concentrations for different periods of time and the effect of sonication was investigated with respect to molecular weight variations and rheological properties. Molar mass, estimated by viscometric measurements, was drastically reduced already after a sonication for a few min. Sonicated samples were used for the preparation of gels in the presence of borate ions. The effect of borax on the new samples was investigated by recording the mechanical spectra and the flow curves. A comparison with the system prepared with the dialysed polymer was also carried out. The anisotropic elongation, observed with tablets of scleroglucan and borax, was remarkably reduced when the sonicated samples were used for the preparation of the gels.     

Controlled Click‐Assembly of Well‐Defined Hetero‐Bifunctional Cubic Silsesquioxanes and Their Application in Targeted Bioimaging

A general procedure for the assembly of hetero‐bifunctional cubic silsesquioxanes with diverse functionality and a perfectly controlled distribution of functional groups on the inorganic framework has been developed. The method is based on a two‐step sequence of mono‐ and hepta‐functionalization through the ligand‐accelerated copper(I)‐catalyzed azide–alkyne cycloaddition of a readily available octaazido cubic silsesquioxane. The stoichiometry of the reactants and the law of binomial distribution essentially determine the selectivity of the key monofunctionalization reaction when a copper catalyst with strong donor ligands is used. The methodology has been applied to the preparation of a set of bifunctional nano‐building‐blocks with orthogonal reactivity for the controlled assembly of precisely defined hybrid nanomaterials and a fluorescent multivalent probe for application in targeted cell‐imaging. The inorganic cage provides an improved photostability to the covalently attached dye as well as a convenient framework for the 3D multivalent display of the pendant epitopes. Thus, fluorescent bioprobes based on well‐defined cubic silsesquioxanes offer interesting advantages over more conventional fully organic analogues and ill‐defined hybrid nanoparticles and promise to become powerful tools for the study of cell biology and for biomedical applications.     

Synthetic Route to a Benzooxazole Derivative with Heparanase Inhibitory Activity

The synthesis of a benzooxazol-5-yl acetic acid derivative (9) with strong heparanase and angiogenesis inhibitory activity, and thus possible commercial interest, is described in detail.     

Investigation of free and conjugated seleno‐amino acids in wheat bran by hydrophilic interaction liquid chromatography with tandem mass spectrometry

An analytical method for determining seleno‐methionine, methyl‐seleno‐cysteine, and seleno‐cystine in wheat bran was developed and validated. Four different extraction procedures were evaluated to simultaneously extract endogenous free and conjugated seleno‐amino acids in wheat bran in order to select the best extraction protocol in terms of seleno amino acid quantitation. The extracted samples were subjected to a clean‐up by a reversed phase/strong cation exchange solid‐phase extraction and analyzed by chiral hydrophilic interaction liquid chromatography‐tandem mass spectrometry. The optimized extraction protocol was employed to validate the methodology. Process efficiency ranged from 58 to 112% and trueness from 73 to 98%. Limit of detection and limit of quantification were lower than 1 ng/g. Four wheat bran samples were analyzed for both total Se and single seleno‐amino acids determination. The results showed that Se‐ seleno‐methyl‐l selenocysteine was the major seleno‐amino acid in wheat bran while seleno‐methionine and seleno‐cysteine were both minor species.